Altering the interface of hydrocarbon-coated surfaces

ABSTRACT

Methods and compositions are provided wherein microorganisms are used to alter the interface of hydrocarbons and hydrocarbon-coated surfaces to increase oil recovery, for improved bioremediation and/or to benefit pipeline maintenance.

This is a divisional application claiming the benefit U.S. applicationSer. No. 13/226,817 filed on Sep. 7, 2011, and U.S. Divisionalapplication Ser. No. 14/467082 filed on Aug. 25, 2014, and U.S.Divisional application Ser. No. 14/083688 filed on Nov. 19, 2013, andU.S. application Ser. No. 12/784,518 filed May 21, 2010, now grantedU.S. patent application Ser. No. 8,658,412B2, and U.S. ProvisionalApplications 61/180,529 and 61/180,445, each filed on May 22, 2009.

FIELD OF INVENTION

This invention relates to the field of environmental microbiology andmodification of heavy crude oil properties using microorganisms. Morespecifically, microorganisms are used to alter the interface betweenhydrocarbons and a surface to increase oil recovery from hydrocarboncoated surfaces.

BACKGROUND OF THE INVENTION

Hydrocarbons in the form of petroleum deposits and oil reservoirs aredistributed worldwide. These oil reservoirs are measured in the hundredsof billions of recoverable barrels. Because heavy crude oil has arelatively high viscosity and may adhere to surfaces, it is essentiallyimmobile and cannot be easily recovered by conventional primary andsecondary means.

Microbial Enhanced Oil Recovery (MEOR) is a methodology for increasingoil recovery by the action of microorganisms (Brown, L. R., Vadie, A. A,Stephen, O. J. SPE 59306, SPE/DOE Improved Oil Recovery Symposium,Oklahoma, Apr. 3-5, 2000). MEOR research and development is an ongoingeffort directed at discovering techniques to use microorganisms tobenefit oil recovery (Sunde. E., Beeder, J., Nilsen, R. K. Torsvik, T.,SPE 24204, SPE/DOE 8th Symposium on enhanced Oil Recovery, Tulsa, Okla.,USA, Apr. 22-24, 1992). An effective MEOR treatment for crude oildesorption and mobilization could utilize microbially derived surfaceactive agents (McInerney, M. J., et al., Development of microorganismswith improved transport and biosurfactant activity for enhanced oilrecovery. DE-FE-02NT15321. DOE, 2003). Few have been identified thathave been shown to alter the surface interaction between hydrocarbonsand rocks, soil, brine, sand or clay to which the hydrocarbons areadhered.

Use of surface active agents or surfactants to increase solubility ofoil through reduction in surface and interfacial tensions is anothertechnique for increasing oil recovery. A wide variety of surfactantsidentified thus far are able to significantly reduce surface andinterfacial tensions at the oil/water and air/water interfaces. Becausesurfactants partition at oil/water interfaces, they are capable ofincreasing the solubility and bioavailability of hydrocarbons (Desai, J.D. and I. M. Banat. Microbial production of surfactants and theircommercial potential. Microbiol. Mol. Biol. Rev., 47-64, 1997 and Banat,I. M. Bioresource Technol. 51: 1-12, 1995 and Kukukina, M. S., et al.Environment International. 31: 155-161, 2005 and Mulligan, C.,Environmental Pollution. 133: 183-198, 2005). Doong and Lei (J.Hazardous Materials. B96: 15-27, 2003), for example, found that theaddition of surfactants to soil environments contaminated withpolyaromatic hydrocarbons increased the mineralization rate of somehydrocarbons (Doong, R and W. Lei, supra). Such surfactants areexpensive and may pose environmental or other equipment issues.

Biosurfactants, (biologically produced surfactants), have helped tosubstantially increase oil recovery from sandstone deposits byincreasing solubility and decreasing viscosity of the oil (Mulligan, C.,supra). Depending on the application, biosurfactants may be preferredsince they are generally more biodegradable and less toxic thansynthetically produced surfactants, and are effective under a broadrange of oil and reservoir conditions. Examples of biosurfactantsinclude glycolipids, lipopeptides and lipoproteins, fatty acids andphospholipids, polymeric compounds, and particulate biosurfactants(Desai, J. D. supra). However, further characterization of productionand use of biosurfactants is needed. Further, there is a need toidentify microorganisms that are able to produce these biosurfactantsunder reservoir conditions or other relevant environmental conditions.

Certain microorganisms have been described as having properties that maybenefit MEOR processes. Certain Shewanella species have been disclosedas useful for remediation of metal contamination (U.S. Pat. No.6,923,914B2), iron containing mixed waste (U.S. Pat. No. 6,719,902B1),manganese contamination (U.S. Pat. No. 6,350,605B1), and otherpollutants with the aid of butane (U.S. Pat. No. 6,245,235B1). InEP1189843, certain Shewanella species were described as being useful forbioremediation of petroleum contaminants aerobically. In addition,Shewanella supplemented with butane was used for reduction of fouling ininjection and recovery wells under aerobic conditions (U.S. Pat. No.6,244,346B1). Other Shewanella species have been described as having theability to produce biofilms (D. Bagge, et al., Appl. Environ. Microbiol.67, 2319-2325. 2001); to sequester gases, in particular CO₂, inunderground geological formations and prevent their release into theatmosphere (see US20060216811A1); and to enhance oil recovery (commonlyowned and co-pending US 2009-0260803 A1). The activity reported by thesemicroorganisms is related to the degradation and transformation ofhydrocarbons and other pollutants and not related to altering theinterfacial boundaries between hydrocarbons and the surfaces to whichthey are bound.

The problem to be solved therefore, relates to the identification ofmicroorganisms that: 1) have the ability to alter the interface betweenhydrocarbons and rock or other surfaces subject to coating by oil; 2)can be inoculated under suitable conditions which effect thesealterations in surface properties; and 3) can be used in acost-efficient way, to improve oil recovery, and benefit bioremediation.

SUMMARY OF THE INVENTION

The methods described herein solve the stated problem above, byidentifying microorganisms that have the ability to alter the interfacebetween hydrocarbons and the surfaces which they coat in order toimprove oil recovery, and benefit bioremediation. The alterations resultin substantial liberation of oil from hydrocarbon-coated surfaces. Inone aspect the microorganisms are Shewanella species that have theability to affect the wettability of the surfaces through microbialaction. In addition, a new isolate of Shewanella sp. has beenidentified.

Accordingly invention provides a method for altering the wettability ofa hydrocarbon coated surface comprising:

-   -   a) providing a hydrocarbon-coated surface;    -   b) providing a medium selected from the group consisting of:        -   i) a cell-containing medium comprising one or more            Shewanella sp.; and        -   ii) a conditioned medium which is substantially cell free            and which has been in contact with one or more Shewanella            sp.;        -   wherein the Shewanella sp. comprises a 16S rDNA comprising            SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:28; and    -   c) contacting said hydrocarbon-coated surface with the medium        of b) wherein the medium alters the wettability of said        hydrocarbon-coated surface.

In another aspect the invention provides a method of oil recoverycomprising:

-   -   a) providing an oil reservoir;    -   b) injecting the oil reservoir with the medium of claim 1; and    -   c) recovering oil from the oil reservoir;        wherein the medium enhances oil recovery.

In another aspect the invention provides a method of treating anenvironmental site comprising:

-   -   a) providing an environmental site comprising hydrocarbon-coated        surfaces;    -   b) contacting the environmental site with the medium of claim 1        wherein the hydrocarbon is released from the site;    -   c) collecting water of the medium and the released hydrocarbon        of (b);    -   d) separating the hydrocarbon and water; and    -   e) making medium of claim 1 using the water of (d) for use in        (b).

In another aspect the invention provides a composition comprising:

-   -   a) at least one Shewanella sp. comprising a 16S rDNA comprising        SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:28; and    -   b) an electron acceptor selected from the group consisting of        nitrate, fumarate, ferric ion, manganese (MnIV) ion and mixtures        thereof.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCES

The invention can be more fully understood from the following detaileddescription, the Figures, and the accompanying sequence descriptions,which form a part of this application.

FIG. 1 shows the relative sand release by strain LH4:18 cultures over aperiod of three weeks. After about 6 days, a 6 mm zone of released sandwas observed in the bottom of the wells for the week old (week 1—solidline) culture and a 3 mm zone was observed for the day-old sample (Day1—dotted line).

FIG. 2 depicts sand/oil release using either an aerobic or an anaerobicculture. PPGAS medium samples (PPGAS), with and without supplements,were used as controls in both aerobic and anaerobic experiments. TheShewanella strain (LH4:18) was used in the experiment. Experiments wereperformed in the absence of lactate and nitrate (L/N) or in theirpresence in both anaerobic and aerobic experiments.

FIG. 3 depicts a comparison of CFU/mL in various samples, expressed asLog 10 (MPN), on the day of the LOOS test set up (Day 3) and therelative sand release for each sample. The symbols designate presence ofthe following ingredients in the growth medium: fumarate (F); nitrate(N); and lactate and nitrate (L/N).

FIG. 4 depicts sand/oil release by Shewanella strain (LH4:18) in thepresence or absence of glucose. One week old cultures were used and theexperiment was continued for 20 days. One set of cultures did notreceive any glucose and another set received 0.5% glucose. Theexperiment was performed for one week in the absence of any heat.

FIG. 5 depicts oil release over time with strain LH4:18 grown indifferent media and supplements for 21 days. Where indicated,supplements were 1% peptone, 1.6 mM MgSO₄, 20 mM KCl, 20 mM NH₄Cl, and20 mM tris base.

FIG. 6 shows the ability of Shewanella strain (LH4:18) to release oil insand/oil release. The strain was tested for 13 days using the SIB mediumeither with peptone or YE added. The controls contained medium but nomicrobes.

FIG. 7 shows sand/oil release using either cell free supernatants of theShewanella strain (LH4:18) cultures or the whole culture. The experimentwas performed for 19 days.

FIG. 8 depicts oil release over time with strain LH4:18 cell freesupernatant (centrifuged compared with centrifuged and filtered) andstrain LH4:18 cell pellet resuspended in fresh medium.

FIG. 9 depicts the results of the ability of strain LH4:18 to releasesand/oil in the presence of other organisms such as Pseudomonas stutzeristrain LH4:15. One set of the experiment was performed measuringsand/oil release by LH4:15, another set used strain LH4:18 and the thirdset used a mixture of both LH4:15 and LH4:18. The experiment wasperformed for 21 days.

FIG. 10 depicts oil release over time with LH4:18 and other Shewanellaspecies (strains EH60:12, EH60:2, and EH60:10). The experiment wasperformed for 19 days.

FIG. 11 depicts oil release over time in a LOOS test in the presence offumarate or nitrate as the electron acceptors. The experiment wasperformed for 14 days with LH4:18 and other Shewanella species purchasedthrough DSMZ (Deutsche Sammlung von Mikrorganismen and Zellkulturen,German Collection of Microorganisms and Cell Cultures).

FIG. 12(A) shows a photomicrograph of untreated oil coated sand.

FIG. 12(B) shows a photomicrograph of oil coated sand treated withShewanella LH4:18. The lines drawn on the picture depict the contactangle between the hydrocarbon and the sand through the oil.

FIG. 13 depicts the level of average residual oil saturation along thesand pack column. The results with control are shown with diamonds andthe results with the inoculated (strain LH4:18) are shown with squares.The amount of residual oil saturation in the control column was 22.5%whereas the residual oil saturation for strain LH4:18 inoculated columnswas 16.1%, indicating that strain LH4:18 was able to reduce residual oilsaturation by approximately 6.5%.

FIG. 14 shows strain and species-specific signature base variations thatoccur in strain L3:3 in the 16S rRNA variable region 3 and SEQ ID NO: 13(bp coordinates 430 to 500) at specific coordinate positions: 438-40,451, 454-57, 466-69, 471, 484-86 and 496.

FIG. 15 shows strain and species-specific signature base variations forbacterial variable region 6 for sequences closely related to Shewanellasp L3:3, e.g., sequences similar to that defined by SEQ ID NO: 14.Strain and species-specific variations occur between base coordinates990 and 1049, specifically at positions: 995-6, 1001-5, 1007, 1012,1014, 1016-18 and 1035.

FIG. 16 shows the Riboprint batch report, 052009, used for comparisonsof Shewanella sp. L3:3 designated riboprint #212-824-S-4 to otherShewanella riboprints available in the Qualicon and DuPont EnvironmentalSciences Riboprint Databases.

FIGS. 17(A) shows a photomicrograph of untreated oil coated sand.

FIG. 17(B) shows that the untreated oil coated sand has a low contactangle (qCA).

FIG. 17(C) shows a photomicrograph of oil coated sand following exposureto strain L3:3.

FIG. 17(D) shows a significant increase in the contact angle of the oilcoated sand (qCB) following exposure to strain L3:3.

FIG. 18 shows dominant and degenerate signature sequences for Shewanellaspecies in rDNA variable regions 2 (A), 5 (B), and 8 (C). The variablepositions are underlined. Alternative nucleotides for each variableposition designation are given in the legend. Shewanella oneidensis MR-1is representative of Shewanella having the-dominant signature sequences.

FIG. 19 is a graph of oil release over time in a LOOS test using strainMPHPW-1 as compared to a medium alone control. The experiment wasperformed for 13 days.

FIG. 20 depicts oil release over time in a LOOS test using strainMPHPW-1 grown with different nutrients added to growth medium. Thenutrients tested were Amberferm 2832, Amberferm 2391, Tastone, andPeptone. The experiment was performed for 13 days.

FIG. 21 shows a schematic diagram of the sandpack apparatus experimentalset-up used to demonstrate oil release. Apparatus parts correspond todesignated numbers as follows: injection brine reservoir (15), pressurepump (16), syringe pump (17), inlet tubing (18), pressure vessel (19),bulkhead pressure fittings (20), pressure gauge (21), flexible tubing(22), sandpack (23), outlet tubing (24), weigh scale (25), absolutepressure gauge (26), differential pressure transducer (27), backpressure regulator (28), jug (29), and weigh scale (30).

FIG. 22 depicts water saturation before and after treatment with strainMPHPW-1. The first part of the curve, labeled 1, is the deoiling curveusing injection brine alone. The second part of the curve, labeled 2, isthe deoiling curve using medium alone without any microorganisms added.The third part of the curve, labeled 3, is the deoiling curve usingmedium plus MPHPW-1. The extrapolated deoiling line from the mediumalone treatment is labeled 4. The extrapolated deoiling line frominjection brine alone treatment is labeled 5.

FIG. 23 depicts sand release over time in a LOOS test using the MPHPW-1inoculum that was used in the sandpack experiment compared to a mediumalone control.

FIG. 24 is Guide Tree, resembling a phylogenetic tree, showing the genusShewanella type strains, Shewanella algae strain BrY, and strainMPHPW-1. The tree was prepared based on a sequence distance method andutilizes Neighbor Joining algorithm of Saitou and Nei from 1987 (infra).

FIG. 25 shows a Riboprint® batch report, 1074, used for comparisons ofstrain Shewanella algae MPHPW-1 to other Shewanella riboprints availablein the Qualicon and DuPont Environmental Sciences Riboprint Databases.

The following DNA sequences conform with 37 C.F.R. 1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures-the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (2009) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions. The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NO:1 is oligonucleotide primer 1492R.

SEQ ID NO:2 is oligonucleotide primer 8F.

SEQ ID NO:3 is16S rDNA from Shewanella sp. L3:3

SEQ ID NO:4 is 16S rDNA from CP000681 Shewanella putrefaciens CN-32.

SEQ ID NO:5 is 16S rDNA from Shewanella putrefaciens LH4:18.

SEQ ID NO:6 is 16S rDNA FJ210800 from Shewanella algae.

SEQ ID NO:7 is 16S rDNA EU563337.1 from Shewanella sp. C13-M.

SEQ ID NO:8 is 16S rDNA EU563345.1 from Shewanella sp. C31.

SEQ ID NO:9 is 16S rDNA DQ164801.1 from Shewanella sp. L-10.

SEQ ID NO:10 is16S rDNA FM210033.2 from Shewanella chilikensis JC5T

SEQ ID NO:11 is 16S rDNA EU721813 from Shewanella uncultured cloneD004024H07.

SEQ ID NO:12 is 16S rDNA EU563338.1from Shewanella sp. C16-M.

SEQ ID NO:13 is the DNA sequence corresponding to prokaryote 16S rRNAvariable region 3 that is signature to Shewanella sp. L3:3 and relatedstrains.

SEQ ID NO:14 is the DNA sequence corresponding to prokaryote 16S rRNAvariable region 6 that is signature to Shewanella sp. L3:3 and relatedstrains.

SEQ ID NO:15 is a partial sequence of the16S rDNA of Shewanella sp.strain EH60:12.

SEQ ID NO:16 is a partial sequence of the16S rDNA of Shewanella sp.strain EH60:10.

SEQ ID NO:17 is a partial sequence of the16S rDNA of Shewanella sp.strain EH60:2.

SEQ ID NO:18 is the Shewanella dominant signature sequence for the 16SrDNA variable region 2.

SEQ ID NO:19 is the Shewanella degenerate signature sequence for the 16SrDNA variable region 2.

SEQ ID NO:20 is the Shewanella dominant signature sequence for the 16SrDNA variable region 5.

SEQ ID NO:21 is the Shewanella degenerate signature sequence for the 16SrDNA variable region 5.

SEQ ID NO:22 is the Shewanella dominant signature sequence for the 16SrDNA variable region 8.

SEQ ID NO:23 is the Shewanella degenerate signature sequence for the 16SrDNA variable region 8.

SEQ ID NO:24 is 16S rDNA from Shewanella algae MPHPW-1

SEQ ID NO:25 is the Shewanella degenerate signature sequence for the 16SrDNA variable region 2 adjusted at position 23 to include the MPHPW-116S rDNA sequence.

SEQ ID NO:26 is 16S rDNA from strain IBI-6P, which is SEQ ID 3 fromUS20100044304.

SEQ ID NO:27 is 16S rDNA from X81621 Shewanella algae BrY.

SEQ ID NO:28 is the Shewanella degenerate signature sequence for the 16SrDNA variable region 2 containing the MPHPW-1 distinguishing C atposition 23.

SEQ ID NO:29 is the DNA sequence corresponding to prokaryote 16S rRNAvariable region 3 that is signature to Shewanella sp. MPHPW-1.

SEQ ID NO:30 is Genbank Accession No. X82131 Shewanella benthica 16SrRNA gene (ATCC 43992) type strain.

SEQ ID NO:31 is Genbank Accession No. X82132 Shewanella hanedai 16S rRNAgene (CIP 103207T) type strain.

SEQ ID NO:32 is Genbank Accession No. X81623 Shewanella putrefaciens 16SrRNA gene type strain.

SEQ ID NO:33 is Genbank Accession No. EF178282 Shewanella haliotisstrain DW01 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:34 is Genbank Accession No. AJ311964 Shewanella denitrificanspartial 16S rRNA gene, strain OS-217 type strain.

SEQ ID NO:35 is Genbank Accession No. EU143361 Shewanella sp. J83 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:36 is Genbank Accession No. AF420312 Shewanella fidelia strainKMM3582T 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:37 is Genbank Accession No. EU290154 Shewanella marinus strainC4 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:38 is Genbank Accession No. U85903 SFU85903 Shewanellafrigidimarina ACAM 591T 16S ribosomal RNA gene, partial sequence typestrain.

SEQ ID NO:39 is Genbank Accession No. U85907 SGU85907 Shewanellagelidimarina ACAM456 16S ribosomal RNA gene, partial sequence typestrain.

SEQ ID NO:40 is Genbank Accession No. AJ300834 Shewanella livingstonis16S rRNA gene, strain LMG19866T type strain.

SEQ ID NO:41 is Genbank Accession No. AM980877 Shewanella vesiculosapartial 16S rRNA gene, type strain M7T type strain.

SEQ ID NO:42 is Genbank Accession No. D21225 VIB16SRRF Shewanellaviolacea gene for 16S rRNA, partial sequence type strain.

SEQ ID NO:43 is Genbank Accession No. FJ589031 Shewanella sp. S4 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:44 is Genbank Accession No. AF005248 Shewanella amazonensis16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:45 is Genbank Accession No. AF005249 Shewanella algae strainATCC 51192 16S ribosomal RNA gene, partial sequence type strain. SEQ IDNO:46 is Genbank Accession No. AF005251 Shewanella oneidensis MR-1 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:47 is Genbank Accession No. AB081757 Shewanella marinintestinagene for 16S rRNA, partial sequence type strain.

SEQ ID NO:48 is Genbank Accession No. AB081760 Shewanella schlegelianagene for 16S rRNA, partial sequence type strain.

SEQ ID NO:49 is Genbank Accession No. AB081762 Shewanella sairae genefor 16S rRNA, partial sequence type strain.

SEQ ID NO:50 is Genbank Accession No. FM203122 Shewanella sp. JC15partial 16S rRNA gene, strain JC15 type strain.

SEQ ID NO:51 is Genbank Accession No. AJ874353 Vibrio natriegens partial16S rRNA gene, strain 01/252.

SEQ ID NO:52 is Genbank Accession No. AF011335 Shewanella pealeana 16Sribosomal RNA gene, complete sequence type strain.

SEQ ID NO:53 is Genbank Accession No. FJ041083 Alteromonadales bacteriumfav-2-10-05 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:54 is Genbank Accession No. AY170366 Shewanella waksmanii 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:55 is Genbank Accession No. AJ000214 Shewanella balticaNCTC10735 16S rRNA gene type strain.

SEQ ID NO:56 is Genbank Accession No. AY190533 Shewanella gaetbuli 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:57 is Genbank Accession No. AB094597 Shewanella surugensisgene for 16S rRNA, partial sequence, strain: c959 type strain.

SEQ ID NO:58 is Genbank Accession No. AB094598 Shewanella kaireiticagene for 16S rRNA, partial sequence, strain: c931 type strain.

SEQ ID NO:59 is Genbank Accession No. AF500075 Shewanella pacifica KMM3597 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:60 is Genbank Accession No. AY326275 Shewanella donghaensisstrain LT17 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:61 is Genbank Accession No. AY351983 Shewanella affinis 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:62 is Genbank Accession No. AJ551089 Shewanella psychrophilapartial 16S rRNA gene, type strain WP2T.

SEQ ID NO:63 is Genbank Accession No. AJ551090 Shewanella piezotoleransWP3 partial 16S rRNA gene, type strain WP3T.

SEQ ID NO:64 is Genbank Accession No. AY445591 Shewanella profundastrain LT13a 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:65 is Genbank Accession No. AJ609571 Shewanella decolorationispartial 16S rRNA gene, type strain CCTCC M 203093T.

SEQ ID NO:66 is Genbank Accession No. AY485224 Shewanella marisflavi 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:67 is Genbank Accession No. AY485225 Shewanella aquimarina 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:68 is Genbank Accession No. AY579749 Shewanella canadensisstrain HAW-EB2 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:69 is Genbank Accession No. AY579750 Shewanella sediminisstrain HAW-EB3 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:70 is Genbank Accession No. AY579751 Shewanella halifaxensisstrain HAW-EB4 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:71 is Genbank Accession No. AY579752 Shewanella atlanticastrain HAW-EB5 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:72 is Genbank Accession No. AF145921 Shewanella sp. KMM329916S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:73 is Genbank Accession No. AY653177 Shewanella colwelliana16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:74 is Genbank Accession No. AB201475 Shewanella abyssi genefor 16S rRNA, partial sequence, strain: c941 type strain.

SEQ ID NO:75 is Genbank Accession No. AB205566 Shewanella hafniensisgene for 16S rRNA, partial sequence, strain: P010 type strain.

SEQ ID NO:76 is Genbank Accession No. AB205570 Shewanellaalgidipiscicola gene for 16S rRNA, partial sequence, strain: S13 typestrain.

SEQ ID NO:77 is Genbank Accession No. AB205571 Shewanellaglacialipiscicola gene for 16S rRNA, partial sequence, strain: T147 typestrain.

SEQ ID NO:78 is Genbank Accession No. AB205576 Shewanella morhuae genefor 16S rRNA, partial sequence, strain: U1417 type strain.

SEQ ID NO:79 is Genbank Accession No. AB204519 Shewanella pneumatophorigene for 16S rRNA, partial sequence type strain.

SEQ ID NO:80 is Genbank Accession No. DQ167234 Shewanella spongiaestrain HJ039 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:81 is Genbank Accession No. DQ180743 Shewanella irciniaestrain UST040317-058 16S ribosomal RNA gene, partial sequence typestrain.

SEQ ID NO:82 is Genbank Accession No. DQ286387 Shewanella loihica strainPV-4 16S ribosomal RNA gene, partial sequence type strain.

SEQ ID NO:83 is Genbank Accession No. AF295592 Shewanella sp. ACEM-9 16Sribosomal RNA gene, partial sequence type strain.

SEQ ID NO:84 is Genbank Accession No. CP000961 Shewanella woodyi strainMS32 =ATCC 51908 REGION: 53358 . . . 54894 type strain.

SEQ ID NO:85 is Genbank Accession No. AP009048 E. coli str. K12 substr.W3110 DNA, complete genome, REGION: 3468481 . . . 3470022.

SEQ ID NO:86 is Genbank Accession No. AE014299 Shewanella oneidensisMR-1 REGION: 46116 . . . 47644.

SEQ ID NO:87 is Genbank Accession No. NC_(—)009438 Shewanellaputrefaciens CN-32 REGION: 38741 . . . 40295.

SEQ ID NO:88 is Genbank Accession No. NC_(—)009052 Shewanella balticaOS155 chromosome, complete genome REGION: 44440 . . . 45976.

SEQ ID NO:89 is Genbank Accession No. CP000821 Shewanella sediminisHAW-EB3, complete genome region 51967-53501.

SEQ ID NO:90 is Genbank Accession No. X81622 Shewanella algae 16S rRNAgene (FeRed).

SEQ ID NO:91 is Genbank Accession No. FJ866783 Shewanella algae strainPSB-05 16S ribosomal RNA gene, partial sequence.

SEQ ID NO:92 is Genbank Accession No. HQ851081 Rhodobacter capsulatusstrain NBY31 16S ribosomal RNA gene, partial sequence.

SEQ ID NO:93 is Genbank Accession No. FJ866781 Shewanella algae strainPSB-04 16S ribosomal RNA gene, partial sequence.

SEQ ID NO:94 is Genbank Accession No. GU223381 Shewanella sp. EM0501 16Sribosomal RNA gene, partial sequence.

SEQ ID NO:95 is Genbank Accession No. HM016084 Shewanella sp. KJW27 16Sribosomal RNA gene, partial sequence.

SEQ ID NO:96 is Genbank Accession No. EU817498 Alishewanella jeotgalistrain MS1 16S ribosomal RNA gene, partial sequence.

SEQ ID NO:97 is Genbank Accession No. X82147 A. rubra 16S rRNA gene(ATCC 29570T).

Applicants have made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Microorganisms for the Purposes of Patent Procedure:

TABLE 1 INFORMATION ON DEPOSITED STRAINS International DepositorIdentification Depository Reference Designation Date of DepositShewanella sp L3:3 ATCC No. PTA-10980 May 19, 2010 Shewanellaputrefaciens ATCC No. PTA-8822 Dec. 4, 2007 LH4:18 Shewanella algaeMPHPW-1 ATCC No. PTA-11920 Jun. 3, 2011

DETAILED DESCRIPTION

Applicants specifically incorporate the entire content of all citedreferences in this disclosure. Unless stated otherwise, all percentages,parts, ratios, etc., are by weight. Trademarks are shown in upper case.Further, when an amount, concentration, or other value or parameter isgiven as either a range, preferred range or a list of upper preferablevalues and lower preferable values, this is to be understood asspecifically disclosing all ranges formed from any pair of any upperrange limit or preferred value and any lower range limit or preferredvalue, regardless of whether ranges are separately disclosed. Where arange of numerical values is recited herein, unless otherwise stated,the range is intended to include the endpoints thereof, and all integersand fractions within the range. It is not intended that the scope of theinvention be limited to the specific values recited when defining arange.

The invention relates to identification of a new strain of Shewanellaisolated from production water samples obtained from an oil reservoirand to methods for altering the interfacial properties of hydrocarboncoated surfaces by contacting these surfaces with these Shewanellamicroorganisms that have the ability to alter the interface between thehydrocarbons and such surfaces. These alterations result in substantialoil liberation from the hydrocarbon-coated surfaces.

The following definitions are provided for the special terms andabbreviations used in this application:

The abbreviation “dNTPs” refers to Deoxyribonucleotide triphosphates.

The abbreviation “ATCC” refers to American Type Culture CollectionInternational Depository, Manassas, Va., USA. “ATCC No.” refers to theaccession number to cultures on deposit with ATCC.

The abbreviation “ASTM” refers to the American Society for Testing andMaterials.

The term “terrestrial subsurface formation” or “subsurface formation”refers to in ground or under ground geological formations and maycomprise elements such as rock, soil, brine, sand, shale, clays andmixtures thereof.

The term “terrestrial surface formation” or “surface formation” refersto above ground geological formations and may comprise elements such asrock, soil, brine, sand, shale, clays and mixtures thereof.

The term “environmental sample” means any sample exposed tohydrocarbons, including a mixture of water and oil. As used hereinenvironmental samples include water and oil samples that compriseindigenous microorganisms useful for phylogenetic mapping of generapresent in a given sampling area.

The term “environmental site” means a site that has been contaminatedwith hydrocarbons and/or other persistent environmental pollutants.Environmental sites may be in surface or subsurface locations.

“Production wells” are wells through which oil is withdrawn from an oilreservoir. An oil reservoir or oil formation is a subsurface body ofrock having sufficient porosity and permeability to store and transmitoil. Production fluid containing a mixture of water and oil is alsorecovered from a production well.

The term “sweep efficiency” refers to the fraction of an oil-bearingstratum that has seen fluid or water passing through it to move oil toproduction wells. One problem that can be encountered with waterfloodingoperations is the relatively poor sweep efficiency of the water, i.e.,the water can channel through certain portions of the reservoir as ittravels from the injection well(s) to the production well(s), therebybypassing other portions of the reservoir. Poor sweep efficiency may bedue, for example, to differences in the mobility of the water versusthat of the oil, and permeability variations within the reservoir whichencourage flow through some portions of the reservoir and not others.

The term “injection water” refers to fluid injected into oil reservoirsfor secondary oil recovery. Injection water may be supplied from anysuitable source, and may include, for example, sea water, brine,production water, water recovered from an underground aquifer, includingthose aquifers in contact with the oil, or surface water from a stream,river, pond or lake. As is known in the art, it may be necessary toremove particulate matter including dust, bits of rock or sand andcorrosion by-products such as rust from the water prior to injectioninto the one or more well bores. Methods to remove such particulatematter include filtration, sedimentation and centrifugation.

The term “irreducible water saturation” is the minimal water saturationthat can be achieved in a porous core plug when flooding with oil tosaturation. It represents the interstitial water content of the matrixwhere the water is never completely displaced by the oil because aminimal amount of water must be retained to satisfy capillary forces.

The term “growing on oil” means the microbial species are capable ofmetabolizing hydrocarbons or other organic components of crude petroleumas a nutrient to support growth.

The term “remediation” refers to the process used to remove hydrocarboncontaminants from an environmental site containing hydrocarbons and/orother persistent environmental pollutants.

The term “bioremediation” refers to the use of microorganisms toremediate or detoxify contaminants form a contaminant-alteredenvironment

“Petroleum” or “oil” is a naturally occurring, flammable liquid found inrock and sand formations in the Earth, which consisting of a complexmixture of hydrocarbons and polycyclic aromatic hydrocarbon of variousmolecular weights, plus other organic compounds.

“Crude oil” refers to the unrefined oil taken from a petroleumreservoir.

“Oil well” and “oil reservoir” may be used herein interchangeably andrefer to a subsurface formation from which oil may be recovered.

“Interface” as used herein refers to the surface of contact or boundarybetween immiscible materials, such as oil and water or a liquid and asolid. As used herein “interfaces” may be between a water layer and anoil layer, a water layer and a solid surface layer, or an oil layer anda solid surface layer.

“Hydrocarbon-coated” as used herein refers to a coating of a hydrocarbonto a solid surface of at least 10% areal coverage.

The term “components of a subsurface formation” refers to rock, soil,brine, sand, shale, clay or mixtures thereof of either subterranean orseabed formations, that have come in contact with one or morehydrocarbon. These components may be part of an oil well or reservoir.At least a portion of the components include some hydrocarbon-coatedsurfaces, including particles with coated surfaces.

“Adhered to” refers to coating or adsorption of a liquid to a solidsurface of at least 10% areal coverage.

“Shewanella species” or “Shewanella sp.” is a bacterial genus that hasbeen established, in part through phylogenetic classification by rDNA.There is at least about 89% sequence identity of 16S rDNA sequencesamong Shewanella species. The 16S rDNA sequences of Shewanella specieshave at least about 89% sequence identity to any of SEQ ID NOs:3-12.Shewanella species have 16S rDNA which has the dominant and degeneratesignature sequences listed respectively of regions 2 (SEQ ID NO:18, 19),5, (SEQ ID NO:20,21) and 8 (SEQ ID NO: 22,23) as shown in FIG. 18. Thedegenerate signature sequence for each region gives the sequence thatdefines Shewanella species, including some position variations as shownin FIG. 18. The dominant signature sequences in FIG. 18 are those withthe variable positions designated as the most frequently foundnucleotides in Shewanella species. Additional Shewanella species havethe degenerate signature sequence for region 2 of SEQ ID NO:28,replacing SEQ ID NO:19. This sequence has a C in position 23 in theregion 2 sequence.

Shewanella are gram negative, gamma-proteobacteria, which have theability to reduce metals and are capable of additionally reducing a widerange of terminal electron acceptors. These microorganisms gain energyto support anaerobic growth by coupling the oxidation of H₂ or organicmatter to the redox transformation of a variety of multivalent metals,which leads to the precipitation, transformation, or dissolution ofminerals.

The abbreviation “rDNA” refers to ribosomal deoxyribonucleic acid genesequence.

The term “rDNA typing” means the process of using the sequence of the16S rDNA gene to obtain the “closest relative” microbial species byhomology to rDNA sequences maintained in several internationaldatabases.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by sequence comparisons. In the art, “identity”also means the degree of sequence relatedness or homology betweenpolynucleotide sequences, as determined by the match between strings ofsuch sequences and their degree of invariance. The term “similarity”refers to how related one nucleotide or protein sequence is to another.The extent of similarity between two sequences is based on the percentof sequence identity and/or conservation. “identity” and “similarity”can be readily calculated by known methods, including but not limited tothose described in “Computational Molecular Biology, Lesk, A. M., ed.Oxford University Press, NY, 1988”; and “Biocomputing: Informatics andGenome Projects, Smith, D. W., ed., Academic Press, NY, 1993”; and“Computer Analysis of Sequence Data, Part I, Griffin, A. M., andGriffin, H. G., eds., Humana Press, NJ, 1994”; and “Sequence Analysis inMolecular Biology, von Heinje, G., ed., Academic Press, 1987”; and“Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., StocktonPress, NY, 1991”. Preferred methods to determine identity are designedto give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs such as sequence analysis software. Typical sequenceanalysis software includes, but is not limited to: the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215, 403-410,1990), DNASTAR (DNASTAR, Inc., Madison, Wis.), and theFASTA program incorporating the Smith-Waterman algorithm (Pearson, W.R., Comput. Methods Genome Res., Proc. Int. Symp, Meeting Date 1992,111-120, Eds: Suhai, Sandor, Plenum Publishing, New York, N.Y., 1994).Within the context of this application, it will be understood that,where sequence analysis software is used for analysis, the results ofthe analysis will be based on the “default values” of the program used,unless otherwise specified. As used herein “default values” will meanany set of values or parameters which originally load with the softwarewhen first initialized.

The term “wetting” refers to the ability of a liquid to maintain contactwith a solid surface, resulting from intermolecular interactions whenthe two are brought together. The degree of wetting (expressed as“wettability”) is determined by a force balance between adhesive andcohesive forces.

“Wetting agent” refers to a chemical such as a surfactant that increasesthe water wettability of a solid or porous surface by changing thehydrophobic surface into one that is more hydrophilic. Wetting agentshelp spread the wetting phase (e.g., water) onto the surface therebymaking the surface more water wet.

“Wettability” refers to the preference of a solid to contact one liquid,known as the wetting phase, rather than another. Solid surfaces can bewater wet, oil wet or intermediate wet. “Water wettability” pertains tothe adhesion of water to the surface of a solid. In water-wetconditions, a thin film of water coats the solid surface, a conditionthat is desirable for efficient oil transport.

The term “adhesive forces” refers to the forces between a liquid andsolid that cause a liquid drop to spread across the surface.

The term “cohesive forces” refers to forces within the liquid that causethe drop to ball up and avoid contact with the surface.

The term “contact angle” is the angle at which a liquid (oil or water)interface meets a solid surface, such as sand or clay. Contact angle isa quantitative measurement of the wetting of a solid by a liquid and isspecific for any given system, and is determined by interactions acrossthree interfaces. The concept is illustrated with a small liquid dropletresting on a flat horizontal solid surface. The shape of the droplet isdetermined by the “Young Relation” (Bico et al., Colloids and SurfacesA: Physicochemical and Engineering Aspects 206 (2002)41-46). Thetheoretical description of contact arises from the consideration of athermodynamic equilibrium between the three phases: the liquid phase ofthe droplet (L), the solid phase of the substrate (S), and the gas/vaporphase of the ambient (V) (which will be a mixture of ambient atmosphereand an equilibrium concentration of the liquid vapor). The V phase couldalso be another (immiscible) liquid phase. At equilibrium, the chemicalpotential in the three phases should be equal. It is convenient to framethe discussion in terms of interfacial energies. The solid-vaporinterfacial energy (see surface energy) is γ_(SV), the solid-liquidinterfacial energy is γ_(SL) L and the liquid-vapor energy (i.e. thesurface tension) is simply γ. The Young equation: 0=γ_(SV)-γ_(SL)-cos θis written such that describes an equilibrium where θ_(C) is theequilibrium contact angle.

“Microbial populations” means one or more populations of microorganismspresent, either in samples obtained from oil wells or in an inoculum forinjection into an oil well or subsurface formation.

“Medium” as used herein means an aqueous milieu which supports thegrowth of organisms. Medium containing Shewanella sp. cells is referredto herein as “cell containing” medium and medium that has been incontact with one or more Shewanella sp. but is a cell free supernatantis referred to herein as “conditioned” medium. Medium will be aqueousbased and may contain various nutrients, buffers, salts, vitamins,co-factors and the like and carbon sources useful for microbial growth.

An “electron acceptor” is a molecular compound that receives or acceptsan electron during cellular respiration. Microorganisms obtain energy togrow by transferring electrons from an “electron donor” to an electronacceptor. During this process, the electron acceptor is reduced and theelectron donor is oxidized. Examples of acceptors include oxygen,nitrate, fumarate, iron (III), manganese (IV), sulfate or carbondioxide. Sugars, low molecular weight organic acids, carbohydrates,fatty acids, hydrogen and crude oil or its components such as petroleumhydrocarbons or polycyclic aromatic hydrocarbons are examples ofcompounds that can act as electron donors.

“Denitrifying” and “denitrification” mean reducing nitrate for use as anelectron acceptor in respiratory energy generation. “Denitrifyingconditions” means conditions where denitrification occurs.

“Inoculating an oil well” means injecting one or more microorganisms ormicrobial populations or a consortium into an oil well or oil reservoirsuch that microorganisms are delivered to the well or reservoir withoutloss of total viability.

The term “simple nitrates” and “simple nitrites” refer to nitrite (NO₂)and nitrate (NO₃).

The term “nutrient supplementation” refers to the addition of nutrientsthat benefit the growth of microorganisms that are capable of usingcrude oil as their main carbon source but grow optimally with othernon-hydrocarbon nutrients, i.e., yeast extract, peptone, succinate,lactate, formate, acetate, propionate, glutamate, glycine, lysine,citrate, glucose, pyruvate and vitamin solutions.

The term “biofilm” means a film or “biomass layer” made up of a matrixof a compact mass of microorganisms consisting of structuralheterogeneity, genetic diversity, complex community interactions, and anextracellular matrix of polymeric substances. Biofilms are oftenembedded in these extracellular polymers, which adhere to surfacessubmerged in, or subjected to, aquatic environment

The term “bacterial” means belonging to the bacteria. Bacteria are anevolutionary domain or kingdom of microbial species separate from otherprokaryotes based on their physiology, morphology and 16S rDNA sequencehomologies.

“Microbial species” means distinct microorganisms identified based ontheir physiology, morphology and phylogenetic characteristics using 16SrDNA sequences. The closest relative microbial species may also bereferred to as a “homolog”.

The term “pure culture” means a culture derived from a single cellisolate of a microbial species. The pure cultures specifically referredto herein include those that are publicly available in a depository.Additional pure cultures are identifiable by the methods describedherein.

The term “simulated injection brine” or “SIB” is a medium compositioncontaining 198 mM NaCl, 1 mM MgCl₂, 1.8 mM CaCl₂, 1.2 mM KCl, 16 mMNaHCO₃, 0.05 mM SrCl₂, 0.13 mM BaCl₂, 0.14 mM LiCl.

The abbreviation “NCBI!” refers to the National Center for BiotechnologyInformation.

A spectrophotometer is a device for measuring light intensity that canmeasure intensity as a function of the color, or more specifically, thewavelength of light. In microbiology, the term “optical density” is aunit-less measure of the transmittance of light at a given wavelengththat uses an empirical relationship that relates the absorption of lightto the properties of the material through which the light is traveling.

The term “MPN” or “most probable number” is a quantitative measurementof the concentration of microbes in a given medium. It is expressed asCFU/ml (colony forming units/ml), log 10(CFU/ml) or log 10(MPN).

The term “hypervariable regions” as used herein refers to sequenceregions in the 16S rRNA gene where the nucleotide sequence is highlyvariable. In most microbes the16S rDNA sequence consists of ninehypervariable regions that demonstrate considerable sequence diversityamong different bacterial genera and species and can be used for genusand species identification

The term “signature sequences” as used herein refers to specificnucleotides at specific16S rRNA encoding gene (rDNA) positions(signature positions), which usually occur within the hypervariableregions, that are distinguishing for microorganisms at different levels.At the signature positions, nucleotides that distinguish between speciesmay be one or more specific base substitutions, insertions or deletions.When taken together, the signature sequences of 16S rDNA are useful fordescribing microbes at the species, strain or isolate level and can beused in the identification of a microbe.

The term “degenerate base position” refers herein to a position in anucleotide sequence that may be more than one choice of nucleotide. Aposition is a “two-fold degenerate” site if only two of four possiblenucleotides may be at that position. A position is a “three-folddegenerate” site if three of four possible nucleotides may be at thatposition. A position is a “four-fold degenerate” site if all fournucleotides may be at that position. “Degeneracy” refers to theexistence of more than one nucleotide in at least one position in aspecified population of sequences having high identity.

The term “degenerate signature sequence” refers to a signature sequencethat has one or more variable position(s) that may include theoccurrence of different bases, base insertions and/or base deletions indifferent individual sequences.

The term “phylogenetic typing” “phylogenetic mapping” or “phylogeneticclassification” may be used interchangeably herein and refer to a formof classification in which microorganisms are grouped according to theirancestral lineage. The methods herein are specifically directed tophylogenetic typing on environmental samples based on 16S Ribosomal DNA(rDNA) sequencing. In this context, approximately 1400 base pair (bp)length of the 16S rDNA gene sequence is generated using 16S rDNAuniversal primers identified herein and compared by sequence homology toa database of microbial rDNA sequences. This comparison is then used tohelp taxonomically classify pure cultures for use in enhanced oilrecovery.

The term “phylogenetics” refers to the field of biology that deals withidentifying and understanding evolutionary relationships betweenorganisms, and in particular molecular phylogenetics uses DNA sequencehomologies in this analysis. In particular, similarities or differencesin 16S rDNA sequences, including signature sequences, identified usingsimilarity algorithms serves to define phylogenetic relationships.

The term “phylogenetic tree” refers to a branched diagram depictingevolutionary relationships among organisms. The phylogenetic tree hereinis based on DNA sequence homologies of 16S rDNAs, including of signaturesequences in the 16S rDNA, and shows relationships of the presentstrains to related reference strains and species.

The term “clade” or “phylogenetic clade” refers to a branch in aphylogenetic tree. A clade includes all of the related organisms thatare located on the branch, based on the chosen branch point. A clade isdefined by a change in one or more signature sequences.

The term “genomovar” is used to describe a sub-species classificationwhich is used when a group of strains of a species are differentiable byDNA sequence, but are phenotypically indistinguishable. Genomovars aredefined and identified by DNA-DNA hybridization and/or by 16S rDNAsignature sequences. This terminology has been used to describePseudomonas stutzeri by Bennasar et al. ((1996) Int. J. of Syst.Bacteriol.46:200-205).

The term “Ribotyping®” refers to fingerprinting of genomic DNArestriction fragments that contain all or part of the rRNA operonencoding for the 5S, 16S and 23S rRNA genes (Webster, John A, 1988. U.S.Pat. No. 4,717,653; Bruce, J. L., Food Techno., (1996), 50: 77-81; andSethi, M. R., Am. Lab. (1997), 5: 31-35). Ribotyping® involvesgeneration of restriction fragments, from microbial chromosomal DNA,which are then separated by electrophoresis, and transferred to a filtermembrane and finally probed with labeled rDNA operon probes. Restrictionfragments that hybridize to the labeled probe produce a distinct labeledpattern or fingerprint/barcode that is unique to a specific microbialstrain. Ribotyping® is performed using the DuPont RIBOPRINTER® system.

The term “Riboprint®” refers to the unique genomic fingerprint of aspecific microbial isolate or strain, generated using the DuPontRIBOPRINTER® system that can be given a unique “Riboprint® identifier”(alphanumeric characters) and stored electronically to be used toidentify the isolate when compared to the database at a later date. Theribotyping® procedure can be entirely performed on the Riboprinter®instrument (DuPont Qualicon, Wilmington, Del.).

The term “Riboprint® batch” refers to comparison alignment of two ormore Riboprints® and is depicted in a report as a pictograph.

The term “type strain” refers to the reference strain recognized in theInternational Journal of Systematic and Evolutionary Microbiology for aparticular species, whose description is used to define and characterizethat species. It is one of the first strains of a species studied and isusually more fully characterized than other strains; however, it doesnot have to be the most representative member. Type strains are usuallydeposited in a national strain collection, such as the ATCC (USA) orDSMZ (German).

The term “reference strain or species” refers to any strain or speciesin the public domain.

The term “standard strain” means a strain which matches the type strainin character and sequence. These strains were used as sequence standardsin defining signature sequences because their genomes have beencompletely sequenced and these sequences scrutinized for accuracy

The abbreviation “LPNSN” refers to the List of Prokaryotic names withStanding in Nomenclature (LPNSN), the official database of acceptedprokaryote names that have conformity within the rules of theInternational Code of Nomenclature of Bacteria and have been validatedby the International Journal of Systematic and EvolutionaryMicrobiology.

The acronym “IPOD” refers to International Patent Organism Depositary,Independent Administrative Agency National Institute of AdvancedIndustrial Science and Technology (AIST Tsukuba Central 6, 1-1, Higashi1-chome, Tsukuba-shi, Ibaraki-ken, Japan), which is a collection ofmicroorganisms.

The acronym “CCUG” refers to the Culture Collection of the University ofGöteborg, Sweden, which is a collection of microorganisms.

The terms “water saturation” and “oil saturation” refer to,respectively, the percentage of void volume occupied by water or oil.

“Void volume” is a measure of empty space in a material that isaccessible by fluids flowing through that material.

Altering Hydrocarbon-Surface Interface

Provided herein are methods for oil recovery and remediation which relyon altering the wettability of hydrocarbon-coated surfaces. Throughaltering wettability, the characteristics of the interface betweenhydrocarbons and a hydrocarbon-coated surface are changed, therebyreleasing the hydrocarbons from the surface. For example, thisalteration may result in the surface having a preference for bindingwater as opposed to oil thereby providing for recovery of the oil morereadily. Changes in the wettability may be monitored by measuringchanges in the contact angle between a hydrocarbon and the surface towhich it is adhered. For example, an increase in the contact angle is anindication of a reduction in the surface energy required to bind the oilto the surface (see Example 8). Thus, an aspect of the present inventionis the discovery that contact between Shewanella sp. or biomoleculesproduced by Shewanella sp., and hydrocarbon coated surfaces producesalterations in the wettability properties of the hydrocarbon coatedsurface such that the surface energy binding the hydrocarbon to thesurface is decreased, (as measured by an increase in the contact angle)resulting in the subsequent release of oil.

Hydrocarbon-coated surfaces may be any hard surface (including one ormore particles) that is coated or contaminated with hydrocarbons with atleast 10% areal coverage by said hydrocarbons. The hydrocarbons may beadhered to said surfaces. Hydrocarbon-coated surfaces may be insubsurface formations, for example in oil reservoirs, and may includerock, soil, brine, sand, clays, shale, and mixtures thereof. Inaddition, hydrocarbon-coated surfaces may include materials that are notsubsurface including rock, clay, soil, sediments, sand, sludge, harbordredge spoils, sediments, refinery wastes, wastewater, sea water, andmixtures thereof. In addition, hydrocarbon-coated surfaces may includeequipment such as pipelines, oil tanks and tankers, and other machinerythat may be contaminated with hydrocarbons.

In the present methods, Shewanella sp. alter the wettability ofhydrocarbon-coated surfaces. Alteration may be by contact with saidmicrobes or by contact with extracellular molecules produced by saidmicrobes, which may include one or more wetting agents. The Shewanellasp. under certain conditions undergo modifications in surface-bound orextracellular molecules. Modifications include changes in thecomposition and/or ratio of the molecules which include but are notlimited to cytochromes, flavins, siderophores, membrane vesicles,glycoproteins, glycolipids, lipoproteins, fimbriae, extracellularpolymeric substances, polysaccharides, monosaccharides, andlipopolysaccharides. These modifications, in turn, alter the waterwettability of a hydrocarbon-coated surface contacted by the alteredShewanella microbe.

Conditions for growth that are suitable for Shewanella species to beused in the present methods are determined by the environment of thetarget hydrocarbon-coated surface, and the conditions for growth of saidspecies in a given environment. Suitable conditions include those thatare favorable to producing changes in the wettability of thehydrocarbon-coated surface. Such suitable conditions may include growthand medium compositions that are beneficial for the production and/ormodification of surface bound or extracellular molecules, especiallythose molecules related to stress, oxygen limitation, redox, and/orelectron transfer which may be wetting agents. Typical growth mediacompositions include enriched media containing diverse nutrient sourcessuch as peptone, yeast extract, or casamino acids, for example. In someaspects the media may be a minimal media such as SL10 or simulatedinjection brine supplemented with an electron donor and electronacceptor. Examples of electron donors include, but are not limited to,lactate and/or acetate. Examples of electron acceptors include but arenot limited to, nitrate, fumarate, pyruvate, ferric ion (Fe (III))and/or manganese ion (Mn (IV). Additional carbon sources may include butare not limited to yeast extract, peptone, pyruvate, glucose, succinate,formate, propionate, glutamate, glycine lysine, oil, and oil components.Oil components may be any of the many components that are present incrude oil. Cultures may be grown aerobically or anaerobically, and maybe grown at a temperature that is similar to that of a target reservoir,typically about 30° C., or in the range of room temperature, +/−5° C. Inaddition, stress conditions may be suitable for growth of the presentstrains. Growth under stress inducing conditions includes, but is notlimited to, switching growth from oxic to anoxic conditions, growthunder population pressure or high density growth, switching electronacceptors, growth at low temperatures, and growth under osmotic stress(such as in high salt).

Cultures of Shewanella species may be used to contact hydrocarbon-coatedsurfaces in the present methods. Alternatively, cells may be removedfrom the cultures and the remaining medium, which has been conditionedby growth of Shewanella species cells, may be used to contacthydrocarbon-coated surfaces in the present methods. It is likely thatconditioned medium contains biosurfactants or other biomolecules thatact as wetting agents and contribute to the alterations in thewettability hydrocarbon coated surfaces.

Multiple cultures of different strains of Shewanella species may be usedin the present methods. Alternatively, multiple strains may be grown inthe same culture that is used in the present methods.

Treating Surface and Subsurface Formations

In the present methods, hydrocarbon-coated surfaces in surface andsubsurface formations are contacted with a Shewanella species culture asa cell containing medium or a conditioned medium. Typically thesubsurface formations will be contained within an oil well site, oftencomprising an injection well site and a production well site.

Application of the medium may include processes such as waterflooding,or the use of a fluid such as an aqueous solution or gas (such as CO₂)or a solvent or a polymer that is injected into the subsurfaceformation. Injection methods are common and well known in the art andany suitable method may be used. For example, Nontechnical guide topetroleum geology, exploration, drilling, and production, 2^(nd)edition. N. J. Hyne, PennWell Corp. Tulsa, Okla., USA, Freethey, G. W.,Naftz, D. L., Rowland, R. C., &Davis, J. A. (2002); Deep aquiferremediation tools: Theory, design, and performance modeling, In: D. L.Naftz, S. J. Morrison, J. A. Davis, & C. C. Fuller (Eds.); and Handbookof groundwater remediation using permeable reactive barriers (pp.133-161), Amsterdam: Academic Press.

Typically the injection site or well will communicate with theproduction well where oil is recovered. The application of the medium(either cell containing or cell free, conditioned) may follow any numberof sequences for the effective production of oil and the various optionswill be readily apparent to the one skilled in the art of oil recovery.

For example treatment of the subsurface formations may include pumpingor adding water containing Shewanella microbes via an injection wellinto an area comprising hydrocarbons (“treatment zone”) and allowingthat water to be produced along with the recovered hydrocarbon at theproduction well. Treatment may also involve pumping water with cell-freemedium produced by conditioning with Shewanella into a treatment zone.Treatment of an oil reservoir also may include pumping water with mediumdown the producer well and into the formation and then back-flowing oiland water out of the producer well (huff and puff). Additionallyreservoir treatment may also include inoculating an injector well thatis in communication with one or more producer wells, and thensubsequently adding an injection water that has been augmented withnutrients either continuously or periodically to promote growth of theShewanella microbes, where oil is recovered at the producer well. Othertreatments may include pumping water containing conditioned medium ontoan environmental site comprising elements as a pile of oil sand or oilshale, collecting the water and released oil, and separating the oilfrom the water. The water may optionally be recycled back to be treatedwith Shewanella sp.

Hydrocarbon-coated surfaces may be contacted with cell containingShewanella sp. medium or conditioned medium alone or with additionalcomponents. Additional components may be provided separately or incompositions with the medium. Components other than cultures may beinjected, pumped, or otherwise applied to an area withhydrocarbon-coated surfaces prior to, together with, or followingcontact with cultures or conditioned medium.

Mixtures of the present one or more Shewanella species and at least oneelectron acceptors provide compositions for use in any oil recovery orclean-up site as listed above. Electron acceptors may include, but arenot limited to, nitrate, fumarate, pyruvate, ferric ion (Fe (III)) ormanganese ion (Mn (IV)). Mixtures of one or more electron acceptor maybe used.

Additional components of the compositions may include at least onecarbon sources, such as but not limited to, lactate, yeast extract,peptone, pyruvate, glucose, succinate, formate, acetate, propionate,glutamate, glycine lysine, oil, and oil components. Oil components maybe any of the many components that are present in crude oil.

The compositions may include other agents or components such as one ormore additional microorganisms, such as bacteria, yeast, or fungus.Particularly useful additional microorganisms are capable of growing onoil under denitrifying conditions. In some embodiments, the additionalagents may be the microorganisms Pseudomonas stutzeri strain LH4:15(ATCC No. PTA-8823), and/or Thauera sp AL9:8, (ATCC No. PTA-9497), whichare described in commonly owned and co-pending US 20090263887 A1, andU.S. Pat. No. 7,708,065. Other agents may also include one or morechemical compounds that are not lethal to microorganisms, but areeffective at degrading or partially degrading hydrocarbons and/or othercontaminants.

Enhanced Oil Recovery From a Reservoir or Oil Well

Enhanced oil recovery in this context may include secondary or tertiaryoil recovery of hydrocarbons from subsurface formations by techniquesthat alter the original properties of hydrocarbon-coated surfaceinterface. Specifically, hydrocarbons that are adhered to surfaceswithin subsurface formations may be substantially liberated by contactwith Shewanella sp. or biomolecules produced by these microorganisms.Typically oil is liberated on an order of about 5, 10, 15, 20, 25, 30,to about 35% of the areal coverage. These methods permit the release ofoil that could not normally be recovered by waterflooding or othertraditional oil recovery techniques.

Bioremediation.

In addition to applications in oil recovery the present Shewanella spmay be useful in effecting the remediation of environmental sitescontaminated with hydrocarbons and other pollutants. Bioremediationstrategies for hydrocarbons depend on their locations in theenvironment. Contamination by hydrocarbon spills can be costly toremediate and cause toxicity to environmental inhabitants. Use ofmicrobial action as described here may provide cost-effective mechanismsfor remediating hydrocarbon contamination especially under circumstancesin which contamination results in hydrocarbon-coated surfaces. Forexample, use of Shewanella sp. and their surface active agents (such aswetting agents) help to increase wettability of soil and solubility ofsoil contaminants through reduction in surface and interfacial tensions.This action liberates the hydrocarbons from the surface of soils andrenders them available for other remediating action, includingdegradation by other microbes. In this context bioremediation may beaccomplished by a combination of microbes including Shewanella sp. inaddition to oil-degrading microorganisms.

Shewanella Species

It has been discovered that the presence of Shewanella species, ormaterials or biomolecules produced by Shewanella, has the effect ofaltering the wettability of a hydrocarbon coated surface. Any and allmembers of the genus Shewanella have this utility.

Shewanella is a bacterial genus that has been established, in partthrough phylogenetic classification by rDNA and is fully described inthe literature (see for example Fredrickson et al., TowardsEnvironmental Systems Biology Of Shewanella, Nature Reviews Microbiology(2008), 6(8), 592-603; Hau et al., Ecology And Biotechnology Of TheGenus Shewanella, Annual Review of Microbiology (2007), 61, 237-258).

It is within the scope of the present invention to classify relevantShewanella on the basis of conserved regions contained in the 16S rDNA.Analysis of the 16 S rDNA from 50 different Shewanella strains revealedthree conserved signature regions each having dominant and degeneratesequences listed respectively: 2 (SEQ ID NO:18, 19), 5, (SEQ ID NO:20,21) and 8 (SEQ ID NO:22, 23) as shown in FIG. 18.

To identify the Shewanella signature sequences, 50 different 16S rDNAsequences of Shewanella strains that are available in the NCBI databasewere aligned. The sequences are from strains that have been classifiedas Shewanella in the International Journal of Systematic andEvolutionary Microbiology. The sequences were aligned using the MegAlignprogram of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Multiple alignment of the sequences was performed usingthe “Clustal method of alignment” (described by Higgins and Sharp,CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci.,8:189-191 (1992)). For multiple alignments, the default valuescorrespond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. In addition tothe Shewanella rDNA sequences, the alignment included 16S rDNA sequencesof E. coli (SEQ ID NO:85), and of microbes closely related toShewanella: Alishewanella jeotgali (SEQ ID NO:96), Pseudoalteromonasrubra (SEQ ID NO:97), and Vibrio natriegenas (SEQ ID NO:51). Throughvisual analysis of the 16S rDNA variable regions 2, 5, and 8, signaturesequences for Shewanella species were identified and are given in FIG.18.

In further analysis of the 16S rDNA sequences of 55 recognized speciesof Shewanella from the List of Prokaryotic names with Standing inNomenclature (LPNSN), as described in Example 26 herein, an additionalbase variation was identified for Shewanella strains that is in position23 of region 2. The sequence for region 2 with the new base variation ofC at position 23 and degeneracy at other positions that are in SEQ IDNO:19 is SEQ ID NO:28, and the degenerate sequence for region 2 thatincludes this variation in position 23 by placing V at this position(V=A/C/G) is SEQ ID NO:25. Thus Shewanella sp. useful in the presentinvention are those that comprise within the 16s rDNA the dominant ordegenerate signature sequences for each of regions 2, 5, and 8 as setforth in SEQ ID NOs:18-23, as well as those with SEQ ID NO:25 and 28 forregion 2 instead of SEQ ID NO:18 or 19. Specifically, Shewanella sp.comprising 16S rDNA comprising SEQ ID NOs:21, 23, and 28 may be used inthe present invention.

Specific strains of Shewanella are disclosed herein that are useful inthe methods of the invention. One such strain is Shewanella putrefaciensstrain LH4:18 which was isolated, identified, and deposited to the ATCCunder the Budapest Treaty as #PTA-8822, as described in commonly ownedand co-pending U.S. Pat. No. 7,776,795. Strain LH4:18 has the 16S rDNAsequence of SEQ ID NO:5.

Examples of additional Shewanella species that may be used include butare not limited to Shewanella frigidimarina (DSM 12253), Shewanellapacifica (DSM 15445), Shewanella profunda (DSM 15900), Shewanellagelidimarina (DSM 12621), and Shewanella baltica (DSM 9439). Thesestrains may be purchased through the German Collection of Microorganismsand Cell Cultures (DSMZ). These and other strains that may be used haveat least about 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5of strain LH4:18.

Additionally useful strains are Shewanella strains EH60:12, EH60:2, andEH60:10, which were identified herein and characterized with partial 16SrDNA sequences of SEQ ID NOs:15, 16, and 17, respectively. Shewanellaspecies include microorganisms having a 16S rDNA sequence with at leastabout 95%, 96%, 97%, 98%, or 99% identity to any one or all of SEQ IDNOs:15-17.

In addition to the known Shewanella sp. described above, a newlyidentified Shewanella sp. which is useful in the present methods wasdisclosed in US Pat. Appl. Pub. #2011-0030956 A1. This new strain isidentified as Shewanella sp. strain L3:3 which has been deposited withthe ATCC under the Budapest Treaty as #PTA-10980. Shewanella sp. strainL3:3 was isolated from an injection water sample obtained from theAlaskan North Slope and has the 16S rDNA sequence of SEQ ID NO:3. Withinthe 16S rDNA sequence are signature sequences that were identified invariable regions 3 and 6 of prokaryote rDNA that have nucleotidesequences of SEQ ID NOs: 13 and 14, respectively. As shown in FIG. 14,the nucleotides at specific positions (with respect to the firstnucleotide of SEQ ID NO:3) 438-40, 451, 454-57, 466-69, 471, 484-86 and496 within SEQ ID NO:13 are different in strain L3:3 from thenucleotides present in the 16S rDNA of Shewanella putrefaciens,Shewanella sp. LH4:18 and Shewanella algae. As shown in FIG. 15, thenucleotides at specific positions (with respect to the first nucleotideof SEQ ID NO:3) 995-6, 1001-05, 1007, 1012, 1014, 1016-1018 and 1035within SEQ ID NO:14 are also different in strain L3:3 from thenucleotides present in the 16S rDNA of Shewanella putrefaciens,Shewanella sp. LH4:18 and Shewanella algae. Shewanella strains foundherein to have the same nucleotides at all of these positions areShewanella sp. C31 (SEQ ID:8), Shewanella sp. L-10(SEQ ID:9), Shewanellachilikensis JC5T (SEQ ID:10), Shewanella sp. C16-M (SEQ ID:12), and aShewanella clone identified as D00402024H07(SEQ ID:11). While having thesignature sequences of SEQ ID NOs:13 and 14, the present Shewanellaspecies that are closely related to the newly identified strain L3:3have at least about 97%, 98% or 99% sequence identity to the DNAsequences for 16S ribosomal RNA of SEQ ID NO:3. In addition, strainsclosely related to strain L3:3 have a Riboprint® pattern identifier of212-824-S-4 as demonstrated in FIG. 16. This Riboprint® pattern wasidentified herein for Shewanella sp. strain L3:3.

Within the 16S rDNA sequence are signature sequences that wereidentified in variable regions 3 and 6 of prokaryote rDNA that havenucleotide sequences of SEQ ID NOs: 13 and 14, respectively. As shown inFIG. 14, the nucleotides at specific positions (with respect to thefirst nucleotide of SEQ ID NO:3) 438-40, 451, 454-57, 466-69, 471,484-86 and 496 within SEQ ID NO:13 are different in strain L3:3 from thenucleotides present in the 16S rDNA of Shewanella putrefaciens,Shewanella sp. LH4:18 and Shewanella algae. As shown in FIG. 15, thenucleotides at specific positions (with respect to the first nucleotideof SEQ ID NO:3) 995-6, 1001-05, 1007, 1012, 1014, 1016-1018 and 1035within SEQ ID NO:14 are also different in strain L3:3 from thenucleotides present in the 16S rDNA of Shewanella putrefaciens,Shewanella sp. LH4:18 and Shewanella algae. Shewanella strains foundherein to have the same nucleotides at all of these positions areShewanella sp. C31, Shewanella sp. L-10, Shewanella chilikensis JC5T,Shewanella sp. C16-M, and a Shewanella clone identified as D00402024H07.While having the signature sequences of SEQ ID NOs:13 and 14, thepresent Shewanella species that are closely related to the newlyidentified strain L3:3 have at least about 97%, 98% or 99% sequenceidentity to the DNA sequences for 16S ribosomal RNA of SEQ ID NO:3.

The present invention provides a newly identified strain of Shewanellawhich is useful in the present methods. This new strain is identified asShewanella algae strain MPHPW-1 which was isolated, identified, anddeposited to the ATCC under the Budapest Treaty as # PTA-11920. The newstrain named MPHPW-1 was isolated from oil well production watercollected from an oil reservoir near Wainwright, Alberta Canada. The 16SrDNA sequence of MPHPW-1 was determined to be SEQ ID NO:24.

Strain MPHPW-1 was identified as a Shewanella strain by using 16S rDNAsequence analysis consistent with the criteria set forth in theInternational Journal of Systematic and Evolutionary Microbiology (B. J.Tindall, R. Rosselló-Mora, H.-J. Busse, W. Ludwig and P. Kämpfer, Int.J. Syst. Evol. Microbiol. 60 (2010), pp. 249-266). A multiple sequencealignment was performed of the MPHPW-1 16S rDNA sequence and the 16SrDNA sequences of the type strains of 55 recognized species ofShewanella from the List of Prokaryotic names with Standing inNomenclature (LPNSN), as well as a few additional representative strainsthat are listed in Table 13. This multiple sequence alignment showedthat among all pairs of the aligned Shewanella type species sequencesthere was at least 90% identity with the MPHPW-1 sequence confirmingthat MPHPW-1 is a Shewanella.

Of the strains in this alignment, MPHPW-1 is most closely related to thethe BrY strain that is classified as a Shewanella algae (Venkateswaran,K, et al. International Journal of Systematic Bacteriology (1999)pp.705-724). This relationship is shown in the phylogenetic tree diagramin FIG. 24, which is described in Example 26 herein. Based on thisrelationship, the MPHPW-1 strain was identified as Shewanella algaeMPHPW-1.

To further characterize strain MPHPW-1, the MPHPW-1 16S rDNA sequencewas aligned with all sequences retrieved in a BLAST search having atleast as high percent identity as the BrY sequence. Each of thesesequences had at least four position differences with the sequence ofMPHPW-1, including nucleotide changes, insertions, and deletions. Thus,based on the 16S rDNA sequence analysis, MPHPW-1 was identified as a newstrain of Shewanella algae. RiboPrint® analysis confirmed that thegenomic sequences surrounding the rDNA operons in strain MPHPW-1 havedifferent genomic structure than those of any strain represented in theRiboPrint® database (7525 patterns contained within DuPont EnvironmentalServices and Qualicon libraries compiled from samples taken from DuPontas well as another 6950 patterns that DuPont Qualicon has supplied fromstandard identified organisms). Shewanella algae strain BrY, whosesequence identity to strain MPHPW-1 is 99.8%, has a RiboPrint® patternthat is similar to that of MPHPW-1, but its pattern is missing the 1 kbband (fragment). Therefore strain MPHPW-1's RiboPrint® is a uniquegenomic identifier of the new Shewanella strain MPHPW-1.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art mayascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, may make various changesand modifications of the invention to adapt it to various usages andconditions.

Additional Abbreviations Used in the Examples

The meaning of abbreviations is as follows: “hr” means hour(s); “mL” or:ml” means millilitre; “° C.” means degrees Celsius; “mg” meansmilligram(s); “mm” means millimeter; “g” means gram(s); “GC” means gaschromatography; “g of oil/g of total fluid” means gram of oil per gramof total fluid; “ppm” means part per million; “mM” means millimolar; “%”means percent; “CFU/mL” means colony forming unit per milliliter; :“LB”means Luria broth medium; “min” means minute(s); “mL/min meansmilliliter per minute; “NIC” means non-inoculated control (negativecontrols in microbial culture experiments); “μg/L” means microgram perliter; “nM” means nanomolar; “μM” means micromolar, “PSIG” meanspounds-force per square inch gauge.

GENERAL METHODS Growth of Microorganisms

Techniques for growth and maintenance of anaerobic cultures aredescribed in “Isolation of Biotechnological Organisms from Nature”,(Labeda, D. P. ed. 117-140, McGraw-Hill Publishers, 1990). When usingnitrate as an electron acceptor in anaerobic cultures, growth ismeasured by nitrate depletion from the growth medium over time. Nitrateis utilized as one of the primary electron acceptors under the growthconditions used herein. The reduction of nitrate to nitrogen has beenpreviously described (Moreno-Vivian, C., et al., J. Bacteriol., 181,6573-6584, 1999). In some cases nitrate reduction processes lead tonitrite accumulation, which is subsequently further reduced to nitrogen.Accumulation of nitrite is therefore also considered evidence for activegrowth and metabolism by microorganisms.

Determination of Viable Cell Titer (Most Probable Number: MPN)

In order to determine viable cell titer, samples from cultures or sandpacks were diluted by 1:10 serial dilution in 8 rows per sample of a 96well plate using standard Miller's Luria Broth or Luria Broth with 3.5%NaCl added. Titration was done using an automated Biomek200 roboticpipettor. Growth was determined by visual turbidity and recorded foreach of 8 rows. The most probable number algorithm of Cochran(Biometrics (1950) 6:105-116) was used to determine the viable cells/mland the 95% confidence limits for this number in the original sample.

The serial dilution method plating was used to determine the bacterialtiter of such cultures. A series of 1:10 dilutions of such samples wasplated and the resulting colonies were counted. The number of colonieson a plate was then multiplied by the dilution factor (the number oftimes that the 1:10 dilution was done) for that plate to obtain thebacterial count in the original sample as CFU/mL.

Ion Chromatography

An ICS2000 chromatography unit (Dionex, Banockburn, Ill.) was used toquantitate nitrate and nitrite ions in growth medium. Ion exchange wasaccomplished on an AS15 anion exchange column using a gradient of 2 to50 mM potassium hydroxide. Standard curves were generated and used forcalibrating nitrate and nitrite concentrations.

Genomic DNA Extractions From Bacterial Cultures

To extract genomic DNA from liquid bacterial cultures, cells wereharvested by centrifugation (10,000 rpm, at room temperature) andresuspended in lysis buffer (100 mM Tris-HCL, 50 mM NaCl, 50 mM EDTA,pH8.0) followed by agitation using a Vortex mixer. Reagents were thenadded to a final concentration of 2.0 mg/mL lysozyme, 10 mg/mL SDS, and10 mg/mL Sarkosyl to lyse the cells. After further mixing with a Vortexmixer, 0.1 mg/mL RNAse and 0.1 mg/mL Proteinase K were added to removeRNA and protein contaminants, and the samples were incubated at 37° C.for 1.0-2.0 hr. Post incubation, the samples were extracted twice withan equal volume of a phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v)and once with chloroform: isoamyl alcohol (24:1). One-tenth volume of5.0 M NaCl and two volumes of 100% ethanol were added to the aqueouslayer, and mixed. The tubes were frozen at −20° C. overnight and thencentrifuged at 15,000×g for 30 min at room temperature to pelletchromosomal DNA. The pellets were washed once with 70% ethanol,centrifuged at 15,000×g for 10 min, dried, resuspended in 100 μL ofde-ionized water and stored at −20° C. An aliquot of the extracted DNAwas visualized on an agarose gel to ascertain the quantity and qualityof the extracted DNA.

Direct Colony rDNA Sequence Analysis

Genomic DNA from bacterial colonies was isolated by diluting bacterialcolonies in 50 μL of water or Tris-HCL buffer pH7-8. Diluted colony DNAswere amplified with Phi 29 DNA polymerase prior to sequencing (GenomiPHIAmplification Kit GE Life Sciences, New Brunswick, N.J.). An aliquot(1.0 μL) of a diluted colony was added to 9.0 μL of the Lysis Reagent(from the GenomiPHI Amplification Kit) and heated to 95° C. for 3 minfollowed by immediate cooling to 4° C. 9.0 μL of Enzyme Buffer and 1.0μL of Phi 29 enzyme were added to each lysed sample followed byincubation at 30° C. for 18 hr. The polymerase was inactivated byheating to 65° C. for 10 min followed by cooling to 4° C.

DNA sequencing reactions were set up as follows: 8.0 μL of GenomiPHIamplified sample were added to 8.0 μL of BigDye v3.1 Sequencing reagent(Applied Biosystems, Foster City, Calif.) followed by 3.0 μL of 10 μMprimers SEQ ID NO: 1 in combination with SEQ ID NO: 2 (prepared by SigmaGenosys, Woodlands, Tex.), 4.0 μL of 5× BigDye Dilution buffer (AppliedBiosystems) and 17 μL Molecular Biology Grade water (Mediatech, Inc.,Herndon, Va.). Sequencing reactions were heated for 3 min at 96° C.followed by 200 thermocycles of (95° C. for 30 sec; 55° C. for 20 sec;60° C. for 2 min) and stored at 4° C. Unincorporated fluorescentlylabeled ddNTPs were removed using Edge Biosystems (Gaithersburg, Md.)clean-up plates. Amplified reactions were pipetted into wells of apre-spun 96 well clean up plate. The plate was centrifuged for 5 min at5,000×g in a Sorvall RT-7 (Sorvall, Newtown, Conn.) at 25° C. Thecleaned up reactions were placed directly onto an Applied Biosystems3730 DNA sequencer and sequenced with automatic base-calling. Each ofthe assembled rDNA sequences was compared to the NCBI rDNA database(˜260,000 rDNA sequences) using the BLAST algorithm (Altschul et al.,supra). The primary hit was used as an identifier of the most closelyrelated known species identification. The initial screen using the rDNAcolony direct sequencing reduced the number of colonies to be carriedthrough further screening by 20 fold.

Automated Ribotyping

Automated ribotyping was used for identification of selected strainswith similar 16S rDNA sequence phylogenetic characteristics (Webster,John A, 1988. U.S. Pat. No. 4,717,653; Bruce, J. L., Food Techno.,(1996), 50: 77-81; and Sethi, M. R., Am. Lab. (1997), 5: 31-35).Ribotyping was performed as recommended by the manufacturer (DuPontQualicon Inc., Wilmington, Del.). For these analyses, one fresh colonywas picked, resuspended in the sample buffer and added to the processingmodule for the heat treatment step at 80° C. for 10 min to inhibitendogenous DNA-degrading enzymes. The temperature was then reduced andlytic enzymes lysostaphin and N-acetyl-muramidase, provided by themanufacturer, were added to the sample. The sample carrier was thenloaded onto the Riboprinter® system with other commercial reagents.Restriction enzyme digestion using EcoRI enzyme, gel electrophoresis andblotting steps were completely automated. Briefly, bacterial DNA wasdigested with the EcoRI restriction enzyme and loaded onto an agarosegel, restriction fragments were separated by electrophoresis and thentransferred to a nylon membrane. After a denaturation step, the nucleicacids were hybridized with a sulfonated DNA probe containing the genesfor the small and large rRNA subunits of E. coli, the 5S, 16S, and 23Sribosomal rRNAs. The hybridized labeled-probe was detected by capturinglight emission from a chemiluminescent substrate with a charge-coupleddevice camera. The output consisted of a densitometry finger scandepicting the specific distribution of the EcoRI restriction fragmentscontaining the genomic rDNA sequences and their molecular weights, whichare particular to the genomic DNA sequence of a specific strainindependent of the 16S rDNA sequence.

Measuring the Potential for Microbes to Release Oil From Sand Particles

In order to screen test cultures for the ability to release oil from anonporous silica medium, a microtiter plate assay to measure the abilityof the microbes to release oil/sand from oil-saturated North Slope sandwas developed. The assay is referred to as the LOOS test (Less Oil OnSand). Autoclaved North Slope sand was dried under vacuum at 160° C. for48 hr. Twenty grams of the dried sand was then mixed with 5 mL ofautoclaved, degassed crude oil. The oil-coated sand was then allowed toage anaerobically at room temperature, in an anaerobic chamber, for atleast a week. Microtiter plate assays were set up and analyzed in ananaerobic chamber. Specifically, 2 mL of test cultures were added intothe wells of a 12-well microtiter plate (Falcon Multiwell 12 wellplates, #353225, Becton Dickinson, Franklin Lakes, N.J.). The controlwells contained 2 mL of the medium alone. Approximately 40 mg ofoil-coated sand was then added to the center of each well. Samples werethen monitored over time for release and accumulation of “free” sandthat collected in the bottom of the wells. Approximate diameters (inmillimeters) of the accumulated total sand released were measured. Ascore of 2 mm and above indicates the microbes' potential to release oilfrom the nonporous silica medium.

Gas Chromatography for Determining Residual Oil on Sand

A gas chromatography (GC) method was developed to analyze the sand fromsandpacks for residual oil. An empirical relationship was determinedbased on the North Slope sand and the intrinsic pore volume of packedsand, e.g., for 240 g of packed sand there is a pore volume of about64mL. Weights of the individual sand samples were obtained and the oilon the sand was extracted with a known amount of toluene. A sample ofthis toluene with extracted oil was then analyzed by GC. A calibrationcurve was generated and used to determine the amount of oil in tolueneon a weight percent basis. This was then multiplied by the total amountof toluene used to extract the oil resulting in the total amount of oilon the sand. This value was then divided by the total sample weight toyield the percent of oil with respect to the total sample weight. Theweight percent of oil of the sample was multiplied by the ratio of theempirically derived characteristic of packed North Slope sand (totalweight of sample after being flooded with brine divided by total sandweight, 1.27). This relationship was equal to the amount of oil on drysand. This value was then multiplied by the ratio of the weight of NorthSlope sand to the weight of fluid trapped in the pore space of the sand,3.75. This resulting value was the residual oil left on the sand inunits of g of oil/g of total fluid in the pore space.

Growth Medium and Growth Protocol

PPGAS medium was used in the following Examples unless stated otherwise.The medium contained: 1.6 mM MgSO₄, 20 mM KCl, 20 mM NH₄Cl, 120 mM Trisbase 0.5% glucose and 1% Bacto peptone. The initial culture was grownaerobically in the medium at 25° C.

Sterile injection brine (SIB) contained: 198 mM NaCl, 1 mM MgCl₂, 1.8 mMCaCl₂, 1.2 mM KCl, 16 mM NaHCO₃, 0.05 mM SrCl₂, 0.13 mM BaCl₂, 0.14 mMLiCl) plus 1% peptone.

The SL10 medium had the following composition summarized in Table 2below:

TABLE 2 Composition of the SL10 Medium Final Chemical Growth componentConcentration Source Nitrogen 18.7 mM NH₄Cl Phosphorus 3.7 mM KH₂PO₄Magnesium 984 μM MgCl₂•6H₂O Calcium 680 μM CaCL₂•2H₂O Sodium chloride172 mM NaCl Trace metals 7.5 μM FeCl₂•4H₂O 12 nM CuCl₂•2H₂O 500 nMMnCL₂•4H₂O 800 nM CoCl₂•6H₂O 500 nM ZnCl₂ 97 nM H₃BO₃ 149 nMNa₂MoO₄•2H₂O 100 nM NiCl₂•6H₂O Selenium-tungstate 22.8 nM Na₂SeO₃•5H₂O24.3 nM Na₂WO₄•2H₂O pH buffer/Bicarbonate 29.7 mM NaHCO₃ Vitamins 100μg/L vitamin B12 80 μg/L p-aminobenzoic acid 20 μg/L D(+)-Biotin 200μg/L nicotinic acid 100 μg/L calcium pantothenate 300 μg/L pyridoxinehydrochloride 200 μg/L thiamine-HCL•2H₂O 50 μg/L Alpha-lipoic acid ThepH of the medium was adjusted to between 7.4-7.8.

Example 1 Comparison of the Ability of Early and Late Stage MicrobialCultures to Release Oil From Sand Particles

To determine whether late stationary phase growth enhances oil release,the oil release activity of an anaerobic overnight culture of strainLH4:18 was compared to that of a one week old culture of the samestrain. A culture was grown initially as described above in PPGASmedium. It was then moved into an anaerobic chamber, was supplementedwith 500 ppm sodium lactate and 1000 ppm sodium nitrate, and divided inhalf. One half was used immediately in an anaerobic LOOS test, describedin General Methods. The other half (Week1 culture) was aged (left for aweek in the anaerobic chamber) and then the LOOS test was performedusing this culture.

FIG. 1 shows the relative sand release by strain LH4:18 cultures over aperiod of three weeks. After about 6 days, a 6 mm zone of released sandwas observed in the bottom of the wells for the week old (week 1)culture and a 3 mm zone was observed for the day-old sample (Day 1).Thus these results indicate that late stationary phase growth culturesmay be more effective in expression of wetting agent molecules in thatthe rate of the sand/oil release was higher for the week old sample andcontinued to increase with time.

Example 2 Demonstration of Oil Release During Both Aerobic and AnaerobicGrowth

To ascertain whether oil release occurs when the assay is performedaerobically versus anaerobically, and whether the addition of lactateand nitrate are beneficial, the following experiment was performed. ALOOS test was set up as described above. A culture of strain LH4:18 wasgrown aerobically overnight at 25° C. in the PPGAS medium. It was thendivided in half. One half was supplemented with 1000 ppm sodium lactateand 2000 ppm sodium nitrate. The other half received no furthersupplements. Each of these cultures was then divided into an aerobic setand an anaerobic set. LOOS tests were set up to compare the samples.PPGAS medium alone samples, with and without the respective supplements,were used as controls.

The results showed that the sand/oil release was relatively the sameirrespective of whether the assay was performed aerobically oranaerobically (FIG. 2). Interestingly, the addition of lactate andnitrate had a detrimental effect on both aerobic and anaerobic cultures.It should be noted, however, that even with the aerobic cultures, oxygencould still be limiting due to the high cell density.

Example 3 Comparison Of Electron Acceptors and Their Effect on OilRelease by Strain LH4:18

A survey of the literature shows that fumarate may act as an efficientterminal electron acceptor (Morris, C. J., et al., Biochem. J., 302:587-593, 1994). In addition, in Shewanella species, certain cell surfaceand respiratory molecules are more abundant in cells grown withfumarate, rather than nitrate or iron citrate, as the terminal electronacceptor (Morris et al., supra). In Example 2, it was demonstrated thatnitrate addition was detrimental in oil/sand release. Fumarate wastherefore tested as an acceptable and possibly more advantageousreplacement in this assay.

A frozen stock culture of strain LH4:18 was diluted 1:100 in SIB plus 1%peptone and placed into an anaerobic chamber. The culture was then splitand sodium nitrate (2000 ppm), both sodium lactate (1000 ppm) and sodiumnitrate (2000 ppm), sodium fumarate (2000 ppm), or both sodium lactateand sodium fumarate supplements were added to different samples. Thecontrol sample contained no additional supplements. Samples were grownanaerobically for 3 days. On the second day, samples were fed again withtheir respective supplements. MPNs were monitored at Day 1 and againafter 3 days of anaerobic growth. On Day 3, a LOOS test was set up andsand/oil release was compared across all samples over time.

FIG. 3 shows a comparison of CFU/mL, expressed as Log 10 (MPN), on theday of the LOOS test set up (Day 3) and the relative sand release foreach sample. The results show that even though growth was relatively thesame across all conditions, the sand/oil release was better for samplescontaining fumarate instead of nitrate as the terminal electronacceptor.

Example 4 Demonstration of the Effect of Various Media Formulations onOil Release by Strain LH4:18

For certain bacterial species, glucose is necessary for the expressionof some surface molecules and surfactants. To determine whether glucosecan improve oil release using strain LH4:18, a LOOS test with thisstrain grown aerobically overnight in PPGAS medium with and withoutglucose was performed. The samples were then placed into an anaerobicchamber and the LOOS test was performed anaerobically as describedabove.

FIG. 4 shows that the sand/oil release response was relatively the samewhether glucose was present or not

In order to determine if the effect of strain LH4:18 on oil release waslimited to a rich medium, the LOOS test response was measured usingdifferent media. Media tested were PPGAS, LB, and supplemented simulatedinjection brine (SIB). SIB was supplemented with 1% peptone and eitherMgSO₄ and KCl, NH₄CL, or Tris. Cultures of strain LH4:18 were grownaerobically overnight. Samples were then placed into an anaerobicchamber and the LOOS test was performed anaerobically.

The simulated injection brine with 1% peptone added worked as well asthe other rich medium formulations as shown in FIG. 5. Strain LH4:18grew relatively the same in each of the media. All cultures exhibitedabout the same sand/oil release response in the LOOS test.

To determine whether yeast extract worked as well as peptone in thesimulated injection brine, these supplements were compared directly in aLOOS assay. Strain LH4:18 was grown aerobically overnight at 25° C. inSIB supplemented with 1% peptone or 1% yeast extract (YE). After 20 hr,the SIB/peptone culture had approximately 4.27E+09 CFU/mL and the SIB/YEculture contained about 9.33E+09 CFU/mL. The samples were then placedinto the anaerobic chamber and the LOOS test was performedanaerobically.

The data in FIG. 6 shows that YE may be substituted for peptone with nodetrimental effect on the oil release response.

Example 5 Demonstration of Oil Release by Culture Supernatant

A number of microbial species release surfactants in their surroundingmedia. To determine whether a wetting agent from strain LH4:18 might bereleased into the surrounding medium, a LOOS test was performed usingboth a whole LH4:18 culture and also the supernatant alone of an LH4:18culture. Strain LH4:18 was grown aerobically overnight at 25° C. in SIBsupplemented with 1% peptone. After 20 hr, the culture containedapproximately 1.49E+09 CFU/mL. The culture was then divided into twoaliquots and one aliquot was centrifuged at 12000×g for 3 min to removethe cells. The supernatant was collected from the centrifuged sample andtransferred into a new tube. Both samples were then placed into ananaerobic chamber and the LOOS test was performed anaerobically asdescribed above.

FIG. 7 shows that the supernatant alone released the sand/oil aseffectively as the whole culture indicating that an agent affecting oilrelease was present in the medium.

While the Example above showed that the supernatant alone released thesand/oil almost as effectively as the whole culture, an experiment wasperformed to determine if oil release ability remained surface bound. Aculture of strain LH4:18 was grown overnight at 25° C. in SIBsupplemented with 1% peptone. The culture was then divided into twoaliquots and half was centrifuged at 12000×g for 3 min to remove thecells. The supernatant was collected from the centrifuged sample andtransferred into a new tube. The pellet was then resuspended in freshmedium. The other half of the overnight culture was also centrifuged andthe supernatant was filtered (Supor, 0.2 μm, Pall Corp., Ann Arbor,Mich.) to remove the microorganisms. The three samples (centrifugedsupernatant, filtered supernatant, and resuspended cells) were thenplaced into the anaerobic chamber and the LOOS test was performedanaerobically. FIG. 8 shows that both supernatant samples released theoil/sand equally well, while oil release by the resuspended cells wasless effective. However the resuspended cells were able to cause someoil release.

Example 6 Effect of Strain LH4:18 in Combination With PseudomonasStutzeri Strain LH4:15 in Oil Release

To determine whether the oil release effected by strain LH4:18 iscompromised by the presence of other microbes, a LOOS test was performedon strain LH4:18 alone and also in the presence of Pseudomonas stutzeriLH4:15 (ATCC No. PTA-8823). Specifically, cultures of strains LH4:15 andLH4:18 were grown separately overnight in the PPGAS medium. Three LOOStests were performed: 1) using strain LH4:15 alone; 2) using strainLH4:18 alone; and 3) using the combined cultures. The results shown inFIG. 9 indicate that the oil release ability of strain LH4:18 was notadversely affected by the presence of the other microorganism.

Example 7 Measuring the Effects of Other Shewanella Species in OilRelease

Additional Shewanella strains had been identified through anaerobicenrichments on oil production fluids, using SL10 medium and Fe(III) asthe electron acceptor. Strains EH60:12, EH60:10, and EH60:2 wereidentified as Shewanella species by their 16S rDNA sequences (SEQ IDNOs:15, 16, and 17, respectively). These strains were grown aerobicallyovernight in the LB medium. A LOOS test was set up on 2 mL of the wholecultures as previously described.

The results in FIG. 10 demonstrate that other Shewanella species (e.g.,strains EH60:12, EH60:2, and EH60:10) were also capable of releasingoil. Results were comparable to those of strain LH4:18.

Other known Shewanella species were purchased through the GermanCollection of Microorganisms and Cell Cultures (DSMZ): Shewanellafrigidimarina (DSM 12253), S. pacifica (DSM 15445), S. profunda (DSM15900), S. gelidimarina (DSM 12621), and S. baltica (DSM 9439). Culturesof each strain were grown aerobically overnight in SIB supplemented with1% peptone. The cultures were then split and 1000 ppm sodium lactate and2000 ppm sodium nitrate, or 1000 ppm sodium lactate and 3715 ppm sodiumfumarate were added. A LOOS test was performed on 2 mL of the culturesas previously described. Samples were not adjusted for growth.

FIG. 11 shows that these known Shewanella species also released oil inthe LOOS assay. As in Example 3, those samples grown in the presence offumarate as the electron acceptor performed better than those grown inthe presence of nitrate.

Example 8 Shewanella Increases the Contact Angle of Oil in Deep SedimentSand

Strain LH4:18 was grown aerobically in PPGAS medium and added to a LOOStest as described above. After approximately two weeks, an aliquot ofthe sand was removed from the bottom of the strain LH4:18 well and wascompared microscopically to oil coated sand from a medium alone controlwell. FIG. 12 shows photomicrographs for comparison. FIG. 12 A shows theuntreated oil coated sand. As indicated by the lines drawn on thepicture, the contact angle between the hydrocarbon and sand is lowindicating that the surface energy encourages the hydrocarbon to coatthe entire mineral grain. The right photomicrograph in FIG. 12B showsthe effect of exposure to strain LH4:18. As indicated by the lines drawnon the picture, the contact angle has increased dramatically indicatinga significant change in the surface energy between the hydrocarbon andthe mineral, and showing substantial liberation of hydrocarbon from thesurface of the sand particle. This is a visual demonstration of changein wettability.

Example 9 Measuring Oil Release From Sandpacks Oil Release Sandpack orCore Flood Assay

The potential application of strain LH4:18 in MEOR treatment wasevaluated using the sandpack technique. This was done with an in-housedeveloped Teflon® shrink-wrapped sandpack apparatus. Using a 0.5 inches(1.27 cm) OD and 7 inches (17.78 cm) long Teflon® heat shrink tube, analuminum inlet fitting with Viton® O-ring was attached to one end of thetube by heat with a heat gun. North Slope sand was added to the columnwhich was vibrated with an engraver to pack down the sand and releasetrapped air. A second aluminum inlet fitting with Viton® O-ring wasattached to the other end of the tube and sealed with heat a gun. Thesandpack was then put in an oven at 275° C. for 7 min to evenly heat andshrink the wrap. The sandpack was removed and allowed to cool to roomtemperature. A second Teflon® heat shrink tube was installed over theoriginal pack and heated in the oven as described above. After thecolumn had cooled, a hose clamp was attached on the pack on the outerwrap over the O-ring and then tightened.

Four sandpack columns were flooded horizontally with three pore volumesof SIB1 low bicarbonate (same as SIB but with 1 mM bicarbonate) at 10mL/min via a syringe pump and a 60 mL (Becton Dickinson, Franklin Lakes,N.J.) sterile plastic polypropylene syringe. All four sandpacks werethen flooded with two pore volumes of anaerobic autoclaved crude oil at0.5 mL/min to achieve irreducible water saturation. The crude oil wasaged on the sand for three weeks before inoculating. For the inoculationculture, strain LH4:18 was grown aerobically overnight in PPGAS medium.The culture was then placed in an anaerobic environment where Na-Lactatewas added to 1000 ppm and Na-Nitrate was added to 2000 ppm. This samplewas anaerobically aged for 5 days before inoculating the sandpacks. Twocolumns were anaerobically inoculated with a sample of strain LH4:18 forone pore volume at 0.4 mL/hr. Two control sandpacks were flooded usinganaerobic SIB1 low bicarbonate using the same inoculation procedure. Thefour sandpacks were then shut-in for incubation with the oil for fivedays. After the shut-in, the columns were produced by flushing withanaerobic sterile SIB low bicarbonate at 0.4 mL/hr for three porevolumes to prepare the production flood.

At the conclusion of the production flood, the 7 inches (17.78 cm) slimtubes were sacrificed into 5 one-inch sections labeled A-E. One inch wasskipped at the beginning and at the exit of the slim tube to avoid edgeeffects during analysis. Sections A, C, and E were analyzed for residualoil saturation on the sand by the GC method described in GeneralMethods.

The results in FIG. 13 show that average residual oil saturation in theuninoculated column was 22.5% whereas the residual oil saturation forstrain LH4:18 inoculated columns was 16.1%, indicating that strainLH4:18 was able to reduce residual oil saturation by approximately 6.5%.

Example 10 Discovery of Oil Recovery Activity in Live Injection WaterSample

To screen enrichment cultures, environmental samples or isolated strainsfor the ability to release oil from a nonporous silica medium, amicrotiter plate assay was developed to measure the ability of microbesto release oil/sand from oil-saturated North Slope sand. North Slopesand was autoclaved and then dried under vacuum at 160° C. for 48 hr and20 g of this dried sand was then mixed with 5 mL of autoclaved, degassedcrude oil obtained from Milne point, North Slope. The oil-coated sandwas then allowed to adsorb to the sand and age anaerobically at roomtemperature for at least a week. Microtiter plate assays were set up ina Coy anaerobic chamber (Coy Laboratories Products, Inc., Grass Lake,Mich.). The assay is referred to as the LOOS test (Liberation of Oil OffSand).

Water samples were obtained from production and injection well heads asmixed oil/water liquids in glass 1.0 L brown bottles, filled to the top,capped and sealed with tape to prevent gas leakage. Gas from inherentanaerobic processes sufficed to maintain anaerobic conditions duringshipment. The bottles were shipped in large plastic coolers filled withice blocks to the testing facilities within 48 hr of sampling.

A sample of non sterile (‘Live’) injection water obtained from AlaskanNorth Slope was used in a LOOS test plus and minus Shewanellaputrefaciens strain LH4:18 (ATCC No. PTA-8822) to determine the efficacyof the Shewanella LH4:18 surface active agent in a background microbialpopulation simulated by the live injection water. Live water wasincluded in the LOOS test as a control. A positive LOOS result wasobtained for live injection+/−LH4:18 microbial treatments. The oil/sandrelease scores obtained from these LOOS tests are given in Table 3.

TABLE 3 Response of Live Injection Water vs. Shewanella LH4:18 in theRelease of Oil from Sand in the LOOS Test Time in days Live 0 3 7 10 1826 Test Injection LH Response as diameter Sample Water 4:18 Nutrients¹of sand released (mm) L1  + − none 0 1 2 4 4 4 L2  + − N 0 1 2 3 3 3L3  + − L/N 0 5 6 6 7 7 L4  + − F 0 2 2 4 5 5 L5  + − L/N 0 2 3 4 6 6L6  + + none 0 4 4 4 5 5 L7  + + N 0 2 4 4 4 4 L8  + + L/N 0 4 5 6 6 6L9  + + F 0 4 5 5 6 6 L10 + + L/N 0 3 5 6 6 6 ¹N = Nitrate (2000 ppm);L/N = Lactate (1000 ppm) plus Nitrate (2000 ppm); F = Fumarate (2000ppm)

The degree of oil release response is measured as the diameter of thesand released from oil. The data demonstrates that test sample L3consisting of live injection water released oil faster than the othersamples. This sample was not inoculated with Shewanella LH4:18. Thistest demonstrates that the live injection water contained an agent oragents that facilitated the release of oil from sand independent ofShewanella sp LH4:18.

Example 11 Isolation and Identification of Shewanella Species in OilReservoir Production Water

Aliquots of the live injection water giving positive oil release resultsin the LOOS test were streaked on LB agar plates (Teknova, Hollister,Calif.) in order to isolate and identify those strain(s) present in liveinjection water capable of oil release. Representative colonies withunique morphologies were isolated from the live injection water testsamples. Samples of these isolated colonies were screened by PCRamplification using direct colony rDNA analysis described in the GeneralMethods using both the reverse PCR primer 1492r (SEQ ID NO:1) andforward PCR primer 8f (SEQ ID NO:2). The resultant rDNA sequence fromeach colony was aligned and matched with the GenBank sequence databasefor phylogenetic strain identification.

One isolate, named L3:3, was identified as having 16S rDNA homology toShewanella sp C16-M. Both L3:3 and C16-M strains as well as four otherreported Shewanella isolates (C31, L31, C13-M and JC5T) have 16S rDNAsequences that are similar to a newly proposed Shewanella species, Sh.chilikensis (K. Sucharita et al, (2009) International Journal ofSystematic and Evolutionary Microbiology 59:3111-3115). The 16S rDNAsequence of L3:3 has 99.9% identity to three of the six rDNA genesequences in the GenBank database that could be classified as Shewanellachilikensis: strain JC5T, strain C16-M, and sequence from a populationstudy designated Shewanella clone D004024H07. Shewanella chilikensisJC5T was isolated from a lake mud environment, Shewanella chilikensisC16-M was isolated from a marine environment and Shewanella cloneD004024H07 was isolated (by DuPont) from environmental samples takenfrom an Alaskan oil well (Pham, V. D, et al., Environ. Microbiol.11:176-187 (2008)).

Strain L3:3 was identified to be Shewanella sp L3:3 and was furthercharacterized by DNA sequence analysis to have signature sequenceswithin Shewanella species rDNA sequences. Specifically, Shewanella spL3:3 was found to have 16S rDNA sequence (SEQ ID NO: 3) and signaturesequences within Shewanella 16S rRNA variable regions 3 and 6 that aredefined in SEQ ID NO:13 (within the prokaryote 16S rRNA variable region3) and SEQ ID NO: 14 (within the prokaryote 16S rRNA variable region 6).These signature sequence regions were discovered when the 16S rDNAsequence profile of Shewanella sp L3:3 was aligned with 42 published 16SrDNA sequences of Shewanella sp., which were pared down to the ninesequences (SEQ ID NO:4 through 12) in FIGS. 1 and 2 for demonstration ofthe variations. Shewanella sp L3:3 full 16S rDNA sequence (SEQ ID NO: 3)was used as the alignment anchor. FIG. 14 shows signature basevariations that occur in L3:3 in the 16S rRNA variable region 3 and SEQID NO: 13 (bp coordinates 430 to 500) at specific coordinate positions:438-40, 451, 454-57, 466-69, 471, 484-86 and 496 and are observed assignature in nature when compared across 16S rDNA of various Shewanellaspecies. A similar observation was made for bacterial variable region 6for sequences closely related to Shewanella sp L3:3, e.g., sequencessimilar to that defined by FIG. 15 and SEQ ID NO: 14 can be found inpublished sequences. Strain variations occur between base coordinates990 and 1049, specifically at positions: 995-6, 1001-5, 1007, 1012,1014, 1016-18 and 1035 as shown in FIG. 2.

In addition to strain L3:3, there are six Shewanella 16S rDNA-likesequences, which were found in sequence databases, that contain thediagnostic signature sequences within variable regions 3 and 6 that aresimilar to those defined by SEQ ID NO: 13 and SEQ ID NO: 14. ThisShewanella group includes: uncultured bacterium clone D004024H07 (NCBIGenBank accession No. gb|EU721813|), Shewanella sp C16-M(gb|EU563338.1|), Shewanella sp. L-10 (gb|DQ164801.1|), Shewanella sp.C31 (gb|EU563345.1|) and Shewanella Sp. JC5T (sp.=chilikensis)(gb|FM210033.2|). Shewanella sp. C13-M (gb|EU563337.1|) does not havethe position 471 nucleotide of the L3:3 diagnostic signature.

All strains were isolated from marine environment, oil fields or thebottom of a lagoon. None of these strains at the time of this inventionwere available from the ATCC or DSMZ public depositories to allow forribotyping® comparisons.

Example 12 Riboprint® Analysis of Strain L3:3

To further characterize Shewanella strain L3:3, preparations of thisstrain were analyzed by Riboprinter®and compared to 7525 patternscontained within DuPont Environmental Services and Qualicon librariescompiled from samples taken all over DuPont as well as another 6950patterns that DuPont Qualicon has supplied from standard identifiedorganisms. Based on the analyses of Riboprint® batch 052009 (FIG. 16),which provides a chromosomal fingerprint of the tested strains, it isclear that the Riboprint® pattern for strain L3:3 (sample 1) constitutesa Riboprint® which is unique when compared against the available DuPontRiboprint® Libraries and is designated as Ribogroup® identifier212-824-S-4. It is probable for various strains to share single similarRiboprint® bands generated by hybridizing the labeled E. coli rDNAoperon probe to each strain's genomic Eco RI fragments, but it is theoverall Riboprint® banding pattern that constitutes identification of agiven strain in a specific Riboprint® or Ribogroup® identifier.

Example 13 Enhanced Oil Release by Strain L3:3

The purified strain L3:3 was tested in a LOOS test designed to identifythe strains' efficacy in altering the surface tension of oil coatedsilica particles. Strain L3:3 clearly contributed to oil release fromsand as compared to the efficiency of oil release by Shewanella strainLH4:18 (ATCC No. PTA-8822) as shown in Table 4. Both strains exhibitedrelease of oil/sand from oil coated particles. The ability to releaseoil/sand was similar when fumarate was the electron acceptor for bothShewanella strains tested (LH4:18 and L3:3), but L3:3 appeared to havegreater release as compared to LH4:18 when nitrate was used as theelectron acceptor.

TABLE 4 LOOS test: Oil/sand Release Response for Purified Cultures ofShewanella strains L3:3 and LH4:18 Time in Days 0 2 5 7 9 12 14 ElectronResponse as diameter Test Strain receptor of sand released (mm) 1 L3:3none 0 4 6 7 7 8 8 2 L3:3 L/N¹ 0 2 5 5 7 7 8 3 L3:3 L/F² 0 3 7 7 7 8 8 4LH4:18 none 0 2 6 7 7 7 7 5 LH4:18 L/N 0 0 3 3 3 4 4 L6 LH4:18 L/F 0 3 67 7 7 7 ¹lactate plus nitrate ²lactate plus fumarate

Example 14 Prophetic Anaerobic Growth of Strain L3:3 on Oil as the SoleCarbon Source

To study growth of strain L3:3 as compared to Shewanella LH4:18,purified isolates are inoculated into 20 mL serum vials containing ˜10mL minimal salts medium (Table 5), 1.6 g/l sodium nitrate and 5.0 mL ofautoclaved crude oil. The medium is deoxygenated by sparging the filledvials with a mixture of nitrogen and carbon dioxide followed byautoclaving. All manipulations of bacteria are done in an anaerobicchamber (Coy Laboratories Products, Inc., Grass Lake, Mich.). Thecultures are incubated at ambient temperatures with moderate shaking(100 rpm) for several weeks to several months and monitored for nitrate,nitrite, visible turbidity and visible oil modifications. When thenitrate is depleted in any culture, sodium nitrate at 50 g/l is added tobring its concentration in the medium up to 0.4 g/l sodium nitrate.

TABLE 5 Minimal salts medium Final Chemical Growth componentconcentration Source Nitrogen 18.7 μM NH₄Cl Phosphorus 3.7 μM KH₂PO₄Magnesium 984 μM MgCl₂•6H₂O Calcium 680 μM CaCL₂•2H₂O Sodium chloride172 mM NaCl Trace metals 670 μM nitrilotriacetic acid 15.1 μM FeCl₂•4H₂O1.2 μM CuCl₂•2H₂O 5.1 μM MnCL₂•4H₂O 12.6 μM CoCl₂•6H₂O 7.3 μM ZnCl₂ 1.6μM H₃BO₃ 0.4 μM Na₂MoO₄•2H₂O 7.6 μM NiCl₂•6H₂O pH buffer (7.5 final) 10mM Hepes Selenium-tungstate 22.8 nM Na₂SeO₃•5H₂O 24.3 nM Na₂WO₄•2H₂OBicarbonate 23.8 nM NaHCO₃ vitamins 100 μg/L vitamin B12 80 μg/Lp-aminobenzoic acid 20 μg/L nicotinic acid 100 μg/L calcium pantothenate300 μg/L pyridoxine hydrochloride 200 μg/L thiamine-HCl•2H₂O 50 μg/Lalpha-lipoic acid Electron acceptor 1.0. g/L NaNO_(3,) Na₂ fumarate orFe(III) Na EDTA The pH of the medium is adjusted to 7.5.Strain L3:3 is expected to show growth via nitrate reduction andturbidity increase under denitrifying conditions as does LH4:18.

Example 15 Anaerobic Growth of Strain L3:3 on Oil as the Sole CarbonSource

Strain L3:3 and Shewanella strain LH4:18 were studied and compared intheir abilities for anaerobic growth on oil as the sole carbon sourceusing different electron acceptors. Shewanella strain LH4:18 has beenshow to grow using nitrate as the electron acceptor in commonly ownedand co-pending US 2009-0260803 A1. Shewanella strains L3:3 and LH4:18were inoculated into 20 mL serum vials containing ˜10 mL SL10 minimalsalts medium (Table 2), supplemented with one of the following electronacceptors: NaNO₃ (2000 ppm), Na₂ fumarate (3500 ppm), or Fe(III) Na EDTA(5000 ppm), and overlayed with 5.0 mL of autoclaved crude oil. LH4-18samples were excluded from NaNO₃ test vials. The medium and crude oilhad been deoxygenated by sparging these reagents with a mixture ofcarbon dioxide and nitrogen (20% and 80%, respectively) followed byautoclaving. All manipulations of bacteria were done in an anaerobicchamber (Coy Laboratories Products, Inc., Grass Lake, Mich.) (gasmixture: 5% hydrogen, 10% carbon dioxide and 85% nitrogen). Replicatetest vials were set up per electron acceptor treatment by L3-3 inoculum.The cultures were incubated at ambient temperature for two weeks. Cellgrowth/titer of the test cultures were analyzed by MPNs.

L3:3 grew anaerobically in oil enrichments where crude oil was providedas the sole carbon source when either NaNO₃, Na₂fumarate, or Fe (III) NaEDTA was provided as the electron acceptor. A table of growth results asanalyzed by cell titers recorded as MPN log 10 is listed below in Table.6. Strain L3:3 grew anaerobically 3 logs to cell titers of ˜10⁵-10⁷cells per mL from starting titers of ˜10³ cells per mL after two weeksincubation time. The change in cell numbers as a result of anaerobicgrowth on oil are listed as the log₁₀ of the MPN recorded for growth±0.5 log. The growth of Shewanella strain L3:3 on the different electronacceptors was comparable to that of Shewanella strain LH4:18. StrainL3:3 grew anaerobically on oil using either NaNO₃, Na₂ fumarate, or Fe(III) Na EDTA as the electron acceptor. Cell titers are presented as theaverage of replicate test vials. Shewanella strain LH4:18 also grew onoil using fumarate or Fe (III) Na EDTA as electron acceptor. Its growthon oil using nitrate as an electron acceptor had been previouslydemonstrated in commonly owned and co-pending US 2009-0260803 A1. BothL3:3 and Shewanella strain LH4:18 grew ˜3 logs to titers of ˜10⁵-10⁷cells per mL from starting titers of 10³ cells per mL anaerobicallyafter two weeks incubation time.

TABLE 6 Anaerobic Growth on oil Delta MPN log10 Electron Acceptor StrainNaNO₃ Na₂ fumarate Fe(III) Na EDTA Shewanella strain 3.0 3.2 2.7 L3:3Shewanella strain n.t. 4.7 2.2 LH4:18 n.t. = not tested

Example 16 Determining the Temperature Limits for Growth of Strain L3:3

To determine the optimal temperature range as well as tolerances forShewanella putrefaciens LH4:18 and Shewanella sp L3:3, these strainswere grown at different test temperatures as given in Table 7.

Inoculums of Shewanella strains LH4:18 and L3:3 were grown overnight (16to18 hours) aerobically in LB medium with shaking at 30-35° C. Theseovernight cultures were grown to visible turbidity and relatively highlevels of cell counts as measured by optical density. Aliquots fromthese starter cultures were then used to seed flasks of 10 mL sterile LBmedia that had been pre-incubated at a specific test temperature overnight. Temperature test cultures were seeded at an optical density ofapproximately 0.1 as measured using a spectrophotometer and visiblelight, wavelength of 600 nm. This constituted a dilution of startercultures approximating between a 1:50 and 1:100 dilution. Growth wasthen measured by tracking the turbidity or optical density of culturesover time. The resulting growth rates determined for strains LH4:18 andL3:3 obtained for the different test temperatures is expressed asdoubling time, the time to double cell number, in units of hours; thesmaller the number the faster the growth rate. At doubling times of <2 hthese strains are presumed to compete successfully with backgroundmicrobial populations in situ. Table 7 shows the results for growthrates at the recorded temperatures. Both Shewanella strains were shownto grow at a rate that would allow them to compete with microbialpopulations in situ for a relatively broad range of environmentaltemperatures.

TABLE 7 Average Recorded Doubling Time for selected temperatures ° C.Doubling times (in hours) Temperature strain 10° C. 16° C. 22° C. 27° C.30° C. 32° C. 35° C. 37° C. 41° C. LH4:18 3.18 1.71 1.02 0.95 0.87 0.79*0.97 1.45 No growth L3:3 n.t. n.t. n.t. n.t. n.t. n.t. n.t. 0.98 0.63.n.t. = not tested

Example 17 Anaerobic Growth of Strain L3:3 in the Presence of Oil UnderDenitrifying Conditions

To demonstrate the ability to grow anaerobically in the presence of oilunder denitrifying conditions, an aliquot (10⁴-10⁵ cells)of each ofShewanella strains LH4:18 and L3:3 was inoculated under anaerobicconditions into 20 mL serum vials containing a 1:2 ratio of minimalsalts medium supplemented with sodium lactate. The media formulationused was designed to promote growth and propagation of Shewanella strainL3:3 as well as the oil release mechanism within a reservoirenvironment. The medium composition for anaerobic growth was as follows:10 mL minimal salts medium (Table 4 minimal salts medium), 1000 ppmsodium lactate and ˜2000 ppm sodium nitrate with 5.0 mL of autoclavedcrude oil. Strain LH4:18 acted as a positive control for anaerobicgrowth under denitrifying conditions containing surface active agent(s).Both the medium and crude oil were deoxygenated by sparging the filledvials with a mixture of nitrogen and carbon dioxide followed byautoclaving. All manipulations of bacteria were done in an anaerobicchamber (Coy Laboratories Products, Inc., Grass Lake, Mich.). Thesecultures were incubated at ambient temperatures for several days andmonitored for nitrate and nitrite levels, for visible turbidity, and forvisible changes to the integrity of the oil phase.

Table 8 shows the results of this growth study in the presence of oil. Apure culture of strain L3:3 showed growth through a reduction in lactateand nitrate levels when grown in the presence of oil. These strains alsoshowed ˜two to three logs growth as indicated by MPN data (Table 8).

TABLE 8 Nitrate Reduction as a Measure of Anaerobic Growth In thepresence of Oil with lactate as the primary carbon source % % nitrateLactate Bacteria 16S Genus reduction reduction MPN time isolate ID inoil in oil log10 (days) NIC¹ n.a. 8% 0% n.t.² 14 L3:3 Shewanella 58% 44%7.0 14 chilikensis, JC5T LH4:18 Shewanella 51% 35% 6.4 14 putrefaciens¹NIC: Non inoculated control ²n.t.: not tested

Example 18 Identification of Electron Acceptors Which Promote OilRelease by Strain L3:3

Different terminal electron acceptors (shown in Table 9) were tested inanaerobic growth of strain L3:3 to determine its ability to grow on arange of terminal electron acceptors including fumarate as well asvarious metal oxides. A mixed culture of LH4:18 and L3:3 was also testedwith nitrate and fumarate. Anaerobic test growths were set up usingminimal salts media (Table 4). 20 mL of minimal salts medium wassupplemented with 1000 ppm sodium lactate, where 2000 ppm sodium nitratewas used as the electron acceptor control. The milli-equivalents of thefollowing electron acceptors were each applied in their respectiveelectron assay sample: fumarate, pyruvate, Fe (III) sodium EDTA,manganese dioxide, and vanadium dioxide. The minimal salts base medium,lactate, and terminal electron acceptor preparations were alldeoxygenated by sparging with a mixture of nitrogen and carbon dioxidefollowed by autoclaving. All manipulations of bacteria were done in ananaerobic chamber (Coy Laboratories Products, Inc., Grass Lake, Mich.).These cultures were incubated at ambient temperature for several daysand monitored for growth by increases in visible turbidity as measuredby OD/MPN or by lactate depletion as measured by IC. Results are shownin Table 9.

TABLE 9 Relative Growth obtained for Strains L3:3 and LH4:18 ondifferent electron acceptors using Lactate as supplemental Carbon SourceIron (Fe(III)) Manganese Vanadium Nitrate Fumarate EDTA Dioxide DioxidePyruvate L3:3 + ++ ++ ++ + ++ L3:3 + + ++ n.t.¹ n.t. n.t. n.t. LH4:18¹n.t. not tested

Example 19 Strain L3:3 Increases the Contact Angle of Oil on DeepSediment Sand

Strain L3:3 was grown aerobically overnight in SIB (Synthetic InjectionBrine; Table 10) plus 1% peptone. Samples were then added into ananaerobic LOOS test, described above, and were supplemented with 1000ppm sodium lactate and 2000 ppm sodium nitrate. After approximately oneweek, an aliquot of the sand was removed from the bottom of the strainL3:3 well and was visualized microscopically. FIG. 17(A) is a typicalimage of untreated oil coated sand. As indicated, the contact angle(qCA) between the hydrocarbon and sand is low—the surface energyencourages the hydrocarbon to coat the entire mineral grain. FIG. 17(B)shows the effect of exposure of oil coated sand to strain L3:3. Thecontact angle (qCB) is increased dramatically indicating a significantchange in the surface energy between the hydrocarbon and the mineral

TABLE 10 Components of SIB1 Minimal Medium (per Liter) and addedelectron acceptor and electron donor NaHCO3 0.138 g CaCl₂*6H₂O 0.39 gMgCl₂*6H₂O 0.220 g KCl 0.090 g NaCl 11.60 g Trace metals (Table 4) 1 mLVitamins (Table 4) 1 mL Na₂HPO₄ 0.015 g (10 ppm PO₄) NH₄Cl 0.029 g (10ppm NH₄) Electron donor added Na-Lactate 0.124 g (124 ppm Na-Lactate)Electron acceptor added Na₂nitrate 0.4 g/400 ppm Adjust pH with HCl orNaOH Filter sterilize

Example 20 Demonstration of Strain L3:3 Oil Release Sandpack or CoreFlood Assay

To test the amount of residual oil left in a sandpack after the oilsoaked sandpack was flooded with a water solution that simulated theinjection brine used in flooding an underground oil reservoir, thesandpack was fabricated as per standard methods described by PetroleumReservoir Rock and Fluid Properties, Abhijit Y. Dandehar, CRC Press(2006). A similar core flood/sandpack apparatus and techniques used tooperate it are also described by Berry et al. (SPE paper number 200056,SPE Reservoir Engineering, November 1991, p 429). The use of a similarapparatus and techniques for testing microbial treatments in a sandpackis described by Saikrishna et al. (SPE paper number 89473, (2004)).

To demonstrate that strain L3:3 is capable of oil release, a L3:3culture was applied to a sandpack saturated with oil in an in-housedeveloped Teflon® shrink-wrapped sandpack apparatus that simulatespacked sand of sandstone. The process described herein was used formaking two column sets, a “control” set and a “test” set, which wasinoculated with L3:3 to test its efficacy to release oil from the sandcolumn. Using a 1.1 inches (2.8 cm) diameter, and 7 inches (17.8 cm)long Teflon heat shrink tube, an aluminum inlet fitting with Viton®O-ring was attached to one end of the tube using a heat gun. AlaskanNorth Slope sand was added to the column which was vibrated with anengraver to pack down the sand and release trapped air. A secondaluminum inlet fitting with

Viton® O-ring was attached to the other end of the tube and sealed withheat a gun. The sandpack was then put in an oven at 275° C. for 7 min toevenly heat and shrink the wrap. The sandpack was removed and allowed tocool to room temperature. A second Teflon® heat shrink tube wasinstalled over the original pack and heated in the oven as describedabove. After the column had cooled, a hose clamp was attached on thepack on the outer wrap over the O-ring and then tightened. For thisdemonstration there were four sandpack columns assembled.

The four sandpack columns were flooded horizontally with three porevolumes of SIB1 Synthetic Injection Brine (Table 10) at 10 mL/min via asyringe pump and a 60 mL (BD) sterile plastic polypropylene syringe. Allfour sandpacks were then flooded with two pore volumes of anaerobicautoclaved crude oil at 0.5 mL/min to achieve irreducible watersaturation. The crude oil was aged on the sand for three weeks prior tobeing inoculated with strain L3:3.

For inoculation, the culture was grown aerobically overnight in PPGASmedia (Table 11). The culture was then placed in an anaerobicenvironment where sodium lactate was added to SIB1 minimal brinesolution to a concentration of 1000 ppm and sodium nitrate was added toa concentration of 2000 ppm. The inoculation sample was thenanaerobically aged in an anaerobic chamber (Coy Laboratories Products,Inc., Grass Lake, Mich.) for 5 days before inoculating the sandpacks.After the aging period, two columns were anaerobically inoculated with asample of Shewanella sp L3:3 for one pore volume at 0.4 mL/hr. Twocontrol sandpacks were flooded using anaerobic SIB1, using the sameinoculation procedure. The four sandpacks were then shut-in forincubation with the oil for five days. After the shut-in, the columnswere then produced for three pore volumes with anaerobic sterile SIB1low bicarbonate at 0.4 mL/hr.

TABLE 11 Components for PPGAS Growth Medium (per Liter) Peptone 10 gMgSO₄ 0.2 g KCl 1.5 g NH₄CL 1.07 g Tris HCL Buffer, pH 7.5 120 mL

At the conclusion of the production flood, the 7 inches long slim tubeswere sacrificed into three 1.9-inch sections labeled A-C. One inch wasskipped at the beginning and at the exit of the slim tube to avoid edgeeffects during analysis. Section “A” came from the front end of thecolumn. Sections A, B and C were analyzed for residual oil saturation onthe sand. The amount of oil on the wet sand from the sacrificed slimtubes for residual oil was measured by GC as described above. This valuewas multiplied by the total amount of toluene used to extract the oilresulting in the total amount of oil on the sand. The value obtained wasthen divided by the total sample weight to yield the percent of oil withrespect to the total sample weight. The weight percent of oil of thesample was then multiplied by the ratio of the empirically derivedcharacteristic of packed North Slope sand (total weight of sample afterbeing flooded with brine divided by total sand weight, 1.27). Thisrelationship is equal to the amount of oil on dry sand. This value wasthen multiplied by the ratio of the weight of the North Slope sand tothe weight of the fluid trapped in the pore space of the sand, 3.75. Theresulting value reflected the residual oil left on the sand in units ofg of oil/g of total fluid in the pore space. As shown in Table 12,residual oil left on the column, in fractions A and C of the testcolumn, were less than the controls confirming that the columnsinoculated with the Shewanella sp. L3:3 released more oil thanuninoculated control columns, with an average of 4.1% decrease inresidual oil remaining on the columns when L3:3 was inoculated on thecolumns.

TABLE 12 Residual oil left on sand along the tube length after floodingwith anaerobic sterile “Brine” Average Percent Residual Oil on SandColumn Fraction Assay Column A C Average. Test columns 12.6% 15.3% 13.9%Control 18.9% 17.1% 18.0% columns

Example 21 Isolation of Strain MPHPW-1

Oil well production water collected from an oil reservoir nearWainwright, Alberta Canada was enriched by supplementing with 1% BactoPeptone (Becton Dickinson, Franklin Lakes, N.J.) and cultured byincubating aerobically overnight at room temperature (21-24° C.) withshaking at 220 rpm. This culture was further enriched during ananaerobic LOOS oil release assay that was run as described in GeneralMethods. An aliquot of the overnight culture received 1000 ppm sodiumlactate and 2000 ppm sodium nitrate. Samples were then run in theanaerobic oil release test for approximately two weeks. Samples frommicrotiter plate wells in which oil was released were then plated ontoLB agar plates (Teknova, Hollister, Calif., USA) to obtain pureisolates. Representative colonies with unique morphologies were streakedagain onto LB plates and one strain was selected for further testing,which was named MPHPW-1.

Example 22 Demonstration of Oil Release Potential of Strain MPHPW-1

To further assess the ability of strain MPHPW-1 to release oil from oilsaturated sand, an oil release test was performed. Briefly, a smallvolume of MPHPW-1 frozen stock was plated onto an LB agar plate andallowed to grow at room temperature. Simulated injection brine plus 1%peptone was inoculated with an individual colony from the plate. Theculture was incubated aerobically overnight at room temperature (21-24°C.) with shaking at 220 rpm and grown to a cell density of approximately1×10⁹ CFU/ml, as determined using the MPN assay described in GeneralMethods. An anaerobic LOOS test was performed as described in GeneralMethods. A control sample contained medium (simulated injection brineplus 1% peptone) alone. Sand/oil release was compared in the MPHPW-1 andcontrol samples over time. The oil release results were that strainMPHPW-1 released oil from a sample of oil soaked sand while the mediumalone control released no oil (FIG. 19).

Example 23 Demonstration of Oil Release by Strain MPHPW-1 Grown in thePresence of Different Nutrients

Strain MPHPW-1 was grown in the presence of different media supplements.MPHPW-1 was inoculated into SHS-10 medium (171 mM NaCl, 0.98 mM MgCl₂,1.4 mM CaCl₂, 0.1 mM KCl, 0.16 mM Na₂SO₄, 16.4 mM NaHCO₃) supplementedwith 1% of Amberferm 2382 (Sensient Flavors, Inc., Harbor Beach, Mich.),Amberferm 2391 (Sensient Flavors, Inc), Tastone (Sensient Flavors,Inc.), or Bacto peptone. These supplements are different sources ofsmall peptides and free amino acids. The cultures were incubatedaerobically overnight at room temperature (21-24° C.) with shaking at220 rpm. An anaerobic LOOS test was performed as described in GeneralMethods. Sand/oil release was compared across all samples over time. Theresults in FIG. 20 showed that strain MPHPW-1 released oil when grown inthe presence of the different supplements.

Example 24 Measuring Oil Release by MPHPW-1 From Sandpacks

The potential application of strain MPHPW-1 in MEOR treatment wasevaluated using a sandpack assay. This was done with an in-housesandpack. A schematic diagram of the sandpack experimental set-up isshown in FIG. 21. All numbers below in bold refer to FIG. 21.

A sample of produced sand that was obtained from the Schrader Bluffformation at the Milne Point Unit of the Alaska North Slope was cleanedby washing with a solvent made up of a 50/50 (volume/volume) mixture ofmethanol and toluene. The solvent was subsequently drained and thenevaporated off the sand to produce clean, dry, flowable sand. This sandwas sieved to remove particles less than one micrometer in size. Thesand was packed tightly into a 20 cm long and 3 cm inner diameter,flexible heat shrinkable tubing (23) and compacted by vibration using alaboratory engraver.

Both ends of the sandpack were capped with compression type fittings toretain the sand mix. Flexible ⅛inch (0.32 cm) tubing capable ofsustaining the pressures used in the test was attached to the fittings.The sandpack was mounted into a pressure vessel (19) with the tubing (18and 24) passing through the ends of the pressure vessel using commonlyavailable bulkhead pressure fittings (20). Additional fittings andtubing were used to connect the inlet of the sandpack to a pressure pump(16), and an injection brine reservoir (15). Low flow rate of aconcentrated solution of nutrients could be pumped using a commonsyringe pump (17) and diluted into the brine being fed from the brinereservoir (15). Other common compression fittings, including elbowunions and tees, and tubing connected the inlet of the sandpack to atransducer that measured the pressure above atmospheric pressure(absolute pressure gauge) (26). The inlet of the sandpack was alsoconnected using the same types of tubing and fittings to the highpressure side of a commonly available differential pressure transducer(27). Fittings and tubing connected the outlet of the sandpack to thelow pressure side of the differential pressure transducer (27) and to aback pressure regulator (28). The produced fluid was collected in a jug(29) and periodically weighed using a weigh scale (30) in order toconfirm the flow rate of the feed pump (16). The pressure vessel (19),the sandpack (23) and the bulkhead fittings (20) were weighed as well,as a means to help determine the amount of oil left in the sandpack. Thesignals from the weigh scales (25, 30), and the differential pressureand the absolute pressure transducers (26, 27) were ported to a computerand these signals were monitored and periodically recorded. The pressurevessel (19) around the sandpack was pressurized using nitrogen portedinto the pressure vessel through flexible tubing (22). The nitrogenpressure was monitored using a pressure gauge (21). The nitrogen was ata pressure of about 110 pounds per square inch (psi) (0.74 mega Pascal)while Brine from the feed reservoir (15) flowed through the sandpack andcame out through the back pressure regulator (28). This operation wasperformed such that the pressure in the sandpack (23) was always 5 to 20psi (0.034-0.137 mega Pascal) below the pressure in the pressure vessel(19).

Throughout the following experimental protocol, the sandpack wasoperated under pressure between 85 to 90 psig so any gas would remaindissolved, thus avoiding air or gas occupying the void volume. Beforebeginning the experiment, the sandpack was conditioned by flowing brinethrough the pack at 4 mL/hour for more than two months.

The sandpack was flooded vertically with three pore volumes of sterilesimulated injection brine containing 40 ppt NaCl at 4 mL/min. Thesandpack was then flooded with two pore volumes of anaerobic autoclavedcrude oil at 4.0 mL/min to achieve irreducible water saturation. Thecrude oil was aged on the sand for one week and then flooded off usingthe same brine as above. This process was repeated three more times toinsure that all hysteresis had been removed from the system and that areproducible level of oil saturation could be regenerated with eachoiling and de-oiling of the system.

When this was accomplished the system was ready for inoculation. For theinoculation culture one pore volume of strain MPHPW-1 was grownaerobically overnight in SHS-10 medium (171 mM NaCl, 0.98 mM MgCl₂, 1.4mM CaCl₂, 0.1 mM KCl, 0.16 mM Na₂SO₄, 16.4 mM NaHCO₃) plus 1% Amberferm2391. Just before inoculation of the sandpack, 1000 ppm Na-Lactate and3715 ppm Na-Fumarate were added to the inoculum. The sandpack column wasinoculated with this MPHPW-1 culture at 4.0 mL/min and was shut-in for 5days. After the shut-in, the column was produced by flushing using thesimulated injection brine with 40 ppt NaCl at 4.0 mL/hr for three porevolumes.

The percent of volume occupied by water, also called water saturation,was calculated during the experiment from the monitored weight of thesandpack. The water saturation calculation was performed as follows: Thedifference in oil and water densities along with the void volume of thesandpack together determine the difference in weight of the sandpack ifthe void volume were all oil or all water. The measured density of theoil used in the experiment was 0.91 g/ml and the density of water is 1.0g/ml.

Water saturation calculations used for FIG. 22:

First the theoretical weight swing on column from 100% water to 100% oilwas calculated as follows:

-   30% void fraction (estimated)-   0.09 g/cc density difference (1 g/cc water, 0.91 g/cc oil)-   116 total volume of sandpack in cc (measured ˜1.1 inches in    diameter×7.4 to 7.5 inches in length; used 1.1 inches diameter, 7.45    inches length for calculation)-   3.1 theoretical weight swing from 100% oil to 100% water

0.30×(0.09×116)=3.1 g. (theoretical weight swing)

-   Oil density was measured; water density is known in the art; 30%    void fraction is known in the art.

Next, Column was flooded with oil to the point at which the weight ofthe apparatus did not change. This corresponded to its irreducible watersaturation; the column weight was measured as −2.702 grams and thisweight was used in subsequent calculations as corresponding to 19% watercontent.

The art and prior work with these types of sand packs has shown us thatthe irreducible water saturation or residual water saturation will bearound 10 to 30%. We chose 19% to use in these calculations. It is notthe absolute value that is important; it is the change in the watersaturation units or % void volume that is important.

Next, the amount of oil on column after SIB treatment was determined(baseline to determine effectiveness of subsequent medium-alone andMPHPW-1 treatments).

Calculation:

(measured weight after oil-saturated column washed with SIB)−(Weightwith 100% SIB)=−1.134 g.

Increased water saturation=(−1.134−(−2.702))13.1×100%=50% increase inwater saturation.

Since the starting water saturation was 19%, then the new watersaturation is 50+19=69%.

The amount of oil on column after medium treatment was determined, andthen medium plus MPHPW-1 (referenced in calculation as test solution,meant to denote separate washes, first with just medium, and then withMPHPW-1 plus medium).

Calculation:

(observed weight of column−(−2.702)/3.1×100%+19%=the water saturationunits plotted in FIG. 22.

This calculated water saturation was graphed in FIG. 22, with the Yaxis, labeled “% void volume occupied by water” corresponding to % watersaturation, and the X axis corresponding to the number of void volumesflooded by each solution. The water saturation percentage is ameasurement of effectiveness of oil removal or recovery.

The results showed that MPHPW-1 treatment increased the average watersaturation by about 15%. In FIG. 22, the first part of the curve,labeled 1, is the deoiling curve using injection brine alone. It showsthat after the column has been saturated with oil and flushed withwater, the water saturation is 69%. The second part of the curve,labeled 2 is the deoiling curve using medium alone without anymicroorganisms added. There appears to be an increased water saturationof about 2% (5) The third part of the curve, labeled 3 is the deoilingcurve using medium plus MPHPW-1. There appears to be an increase inwater saturation of about 15% (4).

Example 25 LOOS Test Confirmation of the Oil Release of the SandpackInoculum

To confirm that the MPHPW-1 inoculum used in the sandpack (Example 24above) released oil from oil saturated sand, a standard LOOS test wasperformed as described in General Methods using SHS-10 plus 1% Amberfermmedium. The effect on oil release of a sample of the inoculum used inExample 24 was compared to the effect of SHS-10 plus 1% Amberferm mediumalone. Results are shown in FIG. 23. The MPHPW-1 sandpack inoculumreleased oil whereas the medium alone did not in the first 12 days ofthe assay.

Example 26 Identification of Strain MPHPW-1 as a New Strain ofShewanella

Strain MPHPW-1 was identified as a strain of Shewanella, most closelyrelated to S. algae strain BrY, by using 16S rDNA sequence analysisconsistent with the criteria set forth in the International Journal ofSystematic and Evolutionary Microbiology (B. J. Tindall, R.Rosselló-Mora, H.-J. Busse, W. Ludwig and P. Kämpfer, Int. J. Syst.Evol. Microbiol. (2010), 60:249-266).

Genomic DNA was isolated from a pure single colony of this strain thatwas isolated as described in Example 21. The universal primers ReversePrimer 1492R (SEQ ID NO:1) and Forward Primer 8F (SEQ ID NO:2) were usedto PCR-amplify a near full length 16S rDNA genomic DNA fragment of about1450 by from this isolate. This amplified fragment was cloned and thensequenced five times in each direction and the raw data was compiled toobtain the final sequence (SEQ ID NO: 24). The resulting consensussequence (SEQ ID NO:24) was queried against the NCBI (The NationalCenter for Biotechnology Information) nucleic acid database, using theBLAST (Basic Local Alignment Search Tool) algorithm. High identitysequence hits were sequences of 16S rDNA from strains of Shewanellaspecies. There were no sequences found having 100% identity to SEQ IDNO:24.

The 16S rDNA sequences of the type strains of the 55 recognized speciesof Shewanella from the List of Prokaryotic names with Standing inNomenclature (LPNSN), as well as a few additional representative strainsthat are listed in Table 13 were downloaded from the NCBI Genbanknucleic acid database. A multiple sequence alignment was performedanchored by the E. coli K12 16S rDNA B sequence, which is recognized asthe standard 16S rDNA for base position assignment (Brosius, Jürge, etal., 1981, J. Mol. Biology 148:107; Woese, C. R. 1987. BacterialEvolution. Microbial. Rev. 51:221), to provide base coordinatepositions. The alignment was performed using the global multiplesequence alignment algorithm from the Clustal series of programs,Clustal W, DNAstar Lasergene Version 8.0.3 MegAlign package, MadisonWis. (Chenna, Ramu, et al., (2003) Nucl. Acids Res. 31:3497). Sequenceswere aligned across their entire length. All sequences used in thealignment were from near full length 16S rDNA sequence, which starts atE. coli coordinate base No. 61 and ends at base coordinate 1460. MPHPW-1coordinates in SEQ ID NO:24 that correspond to the E. coli coordinates61-1460 are MPHPW-1 base coordinates 17 (5′-GTCGA) through 1418(GGGC-3′). Four exceptions with regard to sequence coverage from E. colicoordinates 61 to 1460 were the DNA sequences from the following typestrains, which were not complete at the 5′ or 3′ termini

-   Shewanella atlantica strain HAW-EB5 (CCUG 54554; Genbank:AY579752,    SEQ ID:71) sequence from positions 83 to 1376;-   Shewanella canadensis strain HAW-EB2 (CCUG 54553: Genbank AY579749,    SEQ ID:68) sequence from positions 83 to 1368;-   Shewanella sediminis strain HAW-EB3 (DSM 17055: Genbank CP000821,    SEQ ID:89) sequence from positions 83 to 1363;-   Shewanella profunda strain LT13a (DSM 15900: Genbank AY445591, SEQ    ID:64) sequence from positions 198 to 1531.

The multiple sequence alignment showed that among all pairs of thealigned Shewanella type species sequences, and representative speciessequences that were included, there was at least 90% identity withMPHPW-1 confirming that MPHPW-1 is a strain of Shewanella.

The Guide Tree, which resembles a phylogenetic tree, in FIG. 24 is basedon a sequence distance method and utilizes the Neighbor Joining (NJ)algorithm of Saitou and Nei (Saitou, N. and Nei, M. (1987), Theneighbor-joining method: a new method for reconstructing Guide Trees.Mol. Biol. Evol. 4; 406-425). The NJ method works on a matrix ofdistances between all pairs of sequence to be analyzed. These distancesare related to the degree of divergence between the sequences. The GuideTree is calculated after the sequences are aligned. AlignX displays thecalculated distance values in parenthesis following the molecule namedisplayed on the tree for genus Shewanella strains and strain MPHPW-1based on similarity and differences in 16S rRNA gene sequences wasgenerated from this alignment using bootstrap and Clustal W analysis(Vector NTI AlignX software package 10.3.1 Invitrogen, Carlsbad,Calif.). The software positioned strain MPHPW-1 in a phylogenetic cladeconsisting of four species: Shewanella algae, Shewanella haliotis,Shewanella chilikensis and Shewanella marinus. Shewanella algae strainBrY and strain MPHPW-1 formed a sub-clade within this clade that wasseparate from the other Shewanella algae species of the clade,represented by type strain Shewanella algae OK-1 (ATCC 51192). Based onthe close relationship of MPHPW-1 and Shewanella. algae BrY, MPHPW-1 wasdetermined to belong to the Shewanella algae species. Shewanella sp.strain KJW27 (16S rDNA Accession #HM016084) was also most closelyrelated to S. algae BrY by 16S rDNA sequence analysis and has 2 positiondifferences with the MPHPW-1 16S rDNA. The Shewanella sp. KJW27 strainis slated to be named a new species, Shewanella indica, by theInternational Journal of Systematic and Evolutionary Microbiology(personal communication). The naming of this new species and therelationships that it has with BrY and MPHPW-1 may indicate theassignment of MPHPW-1 as a Shewanella indica once the new naming ispublished.

By analyzing the ClustalW alignment, signature sequence positions in the16S rDNA sequence were identified that could be used to distinguishdifferent Shewanella species and also genomavars within the Shewanellaalgae species. These signature positions are listed in Table 14 withposition coordinate numbers of the E. coli K12 W3110 rrnB allele for 16SrDNA sequence. Table 14 lists the hypervariable region of 16S RNAsequence, where each listed signature sequence position is located.Approximate positions of the hypervariable regions designated bynucleotides of the 16S rDNA sequence from E. coli are:

-   hypervariable region 1 between positions 60 and 99;-   hypervariable region 2 between positions 118 and 290;-   hypervariable region 3 between positions 410 and 520;-   hypervariable region 4 between positions 578 and 760;-   hypervariable region 5 between positions 820 and 888;-   hypervariable region 6 between positions 980 and 1048;-   hypervariable region 7 between positions 1071 and 1179;-   hypervariable region 8 between positions 1215 and 1335;-   hypervariable region 9 between positions 1350 and 1480.

In Table 14, the sequences at the signature positions are given forShewanella algae strains ATCC51192 (16S rDNA SEQ ID NO:45), FeRed (16SrDNA SEQ ID NO:90), and BrY (16S rDNA SEQ ID NO:27), and the closeststrains on the phylogenetic tree (with >98% sequence identity):Shewanella haliotis (16S rDNA SEQ ID NO:33), and Shewanella chilikensis(16S rDNA SEQ ID NO:10). All of these strains have the same signaturesequence at the positions shown in the boxed regions of the table, forexample in hypervariable region 1 at positions 74-84 and 88-98. Thesignature sequences for less closely related strains Shewanellaoneidensis (16S rDNA SEQ ID NO:86), Shewanella colwelliana (16S rDNA SEQID NO:73), and Shewanella amazonensis (16S rDNA SEQ ID NO:44), alsoshown in this table, differ at these positions.

A set of signature sequences that distinguished Shewanella algae BrY andstrain MPHPW-1 from the other Shewanella strains occurred at 15 specificpositions: two in variable region 2 (positions 264 and 278), two invariable region 3 (positions 456-463, 488-491), four in variable region5 (positions 847, 853, 856 and 858), four in variable region 6(positions 1000-1001, 1006-1012, 1017-1023 and 1039-1040) and three invariable region 8 (positions 1243-1245, 1283 and 1292-1294). TheShewanella algae BrY sequence differs from that of MPHPW-1 as follows:mismatches at positions 163 and 170 in the MPHPW-1 sequence (positions203 and 213 using E. coli 16S rDNA position numbers as in Table 14below), and deletions from MPHPW-1 following positions 16, 37, and 1421of the MPHPW-1 sequence.

The sequences with closest identity to the MPHPW-1 16S rDNA sequencefrom the BLAST search, other than S. algae BrY, are listed in Table 15.Due to the sequence identities with other Shewanella strains, one ofthese sequences appear to be misclassified as belonging to a strain ofRhodobacter capsulatus rather than Shewanella. Each of these sequenceshad at least four position differences with the sequence of MPHPW-1,including nucleotide changes, insertions, and deletions. Thus, based onthe 16S rDNA sequence analysis, MPHPW-1 was identified as a new strainof Shewanella algae.

The 16S rDNA sequence of MPHPW-1 fell within the Shewanella degeneratesignature sequences shown in FIG. 18 with an exception at position 23 ofSEQ ID NO:19. This position is number 199 with respect to the E. coli16S rDNA positioning standard. In the MPHPW-1 sequence there is a C atthis position. Thus to incorporate the MPHPW-1 sequence at this positioninto the Shewanella degenerate sequence, the degenerate designation waschanged from R (which includes A or G) to V (which includes A, C, or G).The revised Shewanella degenerate signature sequence for the 16Svariable region 2 is SEQ ID NO:25.

The Shewanella degenerate signature sequence for variable region 2 thatspecifies MPHPW-1 and the other members of the clade to which it belongs(Shewanella algae, Shewanella haliotis, Shewanella chilikensis andShewanella marinus) contains all of the degeneracy in SEQ ID NO:25except C is specified at position 23 of region 2 (SEQ ID NO:28).

TABLE 13 Type strains and representative strains of Shewanella species,with GenBank accession numbers to reference rRNA encoding gene sequencesused in alignment SEQ Accession Strain name and ID Genus and Species No.Deposit Identification NO Shewanella putrefaciens X81623 Hammer 95 =ATCC 32 8071. Shewanella hanedai X82132 281 = ATCC 33224 31 Shewanellabenthica X82131 W 145 = ATCC 43992 30 Shewanella colwelliana AY653177LST-W = ATCC 39565. 73 Shewanella algae AF005249 OK-1 = ATCC 51192 45Shewanella frigidimarina U85903 ACAM 591 = ATCC 38 700753 Shewanellagelidimarina U85907 ACAM 456 = ATCC 39 700752 Shewanella woodyi CP000961MS32 = ATCC 51908 84 Shewanella AF005248 SB2B = ATCC 700329 44amazonensis Shewanella baltica AJ000214 CCUG 39356 = DSM 55 9439Shewanella oneidensis AF005251 MR-1 = ATCC 700550. 46 Shewanellapealeana AF011335 ANG-SQ1 = ATCC 52 700345 Shewanella violacea D21225strain.DSS12 = CIP 42 106290 Shewanella japonica AF145921 ATCC BAA-31672 Shewanella denitrificans AJ311964 OS217 = DSM 15013 34 ShewanellaAJ300834 NF22 = LMG 19866. 40 livingstonensis Shewanella olleyanaAF295592 ACEM 9 = LMG 21437 83 Shewanella fidelis AF420312 ATCC BAA-31836 Shewanella AB081757 IK-1 = JCM 11558 47 marinintestina Shewanellasairae AB081762 SM2-1 = JCM 11563. 49 Shewanella AB081760 HRKA1 = JCM11561 48 schlegeliana Shewanella waksmanii AY170366 ATCC BAA-643 54Shewanella affinis AY351983 ATCC BAA-642 61 Shewanella aquimarinaAY485225 SW-120 = JCM 12193 67 Shewanella gaetbuli AY190533 TF-27 = JCM11814 56 Shewanella marisflavi AY485224 SW-117 = JCM 12192 66 Shewanellapacifica AF500075 R10SW1 = DSM 15445 59 Shewanella profunda AY445591LT13a = DSM 15900 64 Shewanella AJ609571 S12 = JCM 21555 65decolorationis Shewanella AB204519 SCRC-2738 = JCM 79 pneumatophori13187. Shewanella sediminis CP000821 HAW-EB3 = DSM 89 17055 Shewanellaabyssi AB201475 c941 = DSM 17171 74 Shewanella hafniensis AB205566 P010= ATCC BAA- 75 1207 Shewanella halifaxensis AY579751 HAW-EB4 = DSM 7017350 Shewanella irciniae DQ180743 UST040317-058 = JCM 81 13528Shewanella kaireitica AB094598 c931 = DSM 17170 58 Shewanella loihicaDQ286387 PV-4 = ATCC BAA- 82 1088 Shewanella morhuae AB205576 U1417 =ATCC BAA- 78 1205 Shewanella spongiae DQ167234 HJ039 = JCM 13830 80Shewanella surugensis AB094597 c959 = DSM 17177 57 Shewanella AB205570S13 = LMG 23746 76 algidipiscicola Shewanella atlantica AY579752 HAW-EB5= CCUG 71 54554 Shewanella canadensis AY579749 HAW-EB2 = CCUG 68 54553Shewanella AY326275 LT17 = JCM 12524 60 donghaensis Shewanella AB205571T147 = LMG 23744 77 glacialipiscicola Shewanella haliotis EF178282 DW01= JCM 14758. 33 Shewanella AJ551090 WP3 = JCM 13877. 63 piezotoleransShewanella AJ551089 WP2 = JCM 13876. 62 psychrophila Shewanella basaltisEU143361 J83 = JCM 14937 35 Shewanella chilikensis FM210033 JC5 = CCUG57101 51 Shewanella marina EU290154 C4 = JCM 15074 37 Shewanellavesiculosa AM980877 M7 = CECT 7339 41 Shewanella corallii FJ041083fav-2-10-05 = DSM 53 21332 Shewanella fodinae FM203122 JC15 = CCUG 5710250 Shewanella FJ589031 S4 = JCM 16212. 43 xiamenensis Shewanella algaeX81621 BrY = ATCC 51181 27 Shewanella baltica NC_009052 OS155 = ATCCBAA- 88 1091 Shewanella putrefaciens NC_009438 CN-32 = ATCC BAA- 87Error! 453 or ATCC BA-1097 Bookmark not defined.

TABLE 14Sequences that are underlined and bold are unique signatures different from theShewanella consensus. Differences between MPHPW-1 and BrY are italicized forMPHPW-1. SEQ ID NOs are in parentheses. Bacterial E.coli K12 Hyper- rDNAShewanella Shewanella Shewanella Shewanella Strain variable coordinatehaliotis algae ATCC algae algae BrY MPHPW-1 Regions No. DW01 (33)51192 (45) FeRed (90) (27) (24) 1 75-84 ATTTCAAA ATTTCAAA ATTTCAAAATTTCAAA

- AG AG AG AG

1 88-98 TTTGAAGAAG TTTGAAGAAG TTTGAAGAAG TTTGAAGA

TGA TGA TGA TGA

2 206 A C C C

2 213 T G G G

2 223-225 TGA TGA TGA TGA TGA 2 230-232 AGG AGG AGG AGG AGG 2 253 A A AA A 2 264 T A A T T 2 278 A G G A A 2 293 G G G G G 2 306-307 AT AT ATAT AT 2 379-381 GGA GGA GGA GGA GGA 2 384 C C C C C 3 456-463 G T GTAAGT TT GTAA GT TT GTAA GT G T GTAA GT G T GTAA GT 3 473-477 TTAC A T TTACA T TTAC A T TTAC A T TTAC A T 3 488-491 C T C G TT

G C T C G C T C G C T C G 3 513 C C C C C 4 539 GA GA GA GA GA 4 546 G GG G G 4 552 T T T T T 4 578 G G G G G 4 590 T T T T T 4 632 C C C C C 4646-649 G C AA G C AA G C AA G C AA G C AA 4 679 C C C C C 4 711 G G G GG 4 743-748 ACAAAG ACAAAG ACAAAG ACAAAG ACAAAG 4 760-763 GGCA GGCA GGCAGGCA GGCA 5 832 G G G G G 5 847 C G C C C 5 853-858 GCTCTC T CT T T AGCTCTC GCTCTC GCTCTC 6 1000-1001 AGA A C A A C A AGA AGA 6 1006-1012 TCT GGT AG C T TTTC AG C T TTTC AG CTTTCCAG C T TT C C AG 6 1017-1023 TACC T CA TGAAT T G TGAAT T G TG G AT T G TG G AT T G 6 1038-40   TGT TGTTGT T C T T C T 6 1060-1063 TGTC TGTC TGTC TGTC TGTC 7 1115 C C C C C 71124 C C C C C 7 1133-34   GG GG GG GG GG 7 1138 A A A A A 7 1140-1141CC CC CC CC CC 7 1157-1160 CTTT CTTT CTTT CTTT CTTT 8 1243-1246 TCAGTCAG TCAG T C G G T C G G 8 1260 G G G G G 8 1273-74   TG TG TG TG TG 81283 C C C T T 8 1292-4     T GG T GG T GG C GG C GG 8 1308 T T T T T 81328 A A A A A 9 1354-1356 TGG TGG TGG TGG TGG 9 1366-1368 CCA CCA CCACCA CCA 9 1401-1402 CC CC CC CC CC 9 1429-30   GG GG GG GG GG 91438-1440 AGA AGA AGA AGA AGA Bacterial Hyper- Shewanella ShewanellaShewanella Shewanella variable chilikensis oneidenses colwellianaamazonensis Regions JC5 (10) MR-1 (86) LST-W (73) SB2B (44) 1 ATTTCAAAACACAAGT AGGATT- GGGAAGAT AG GA TAG AG 1 TTTGAAGA CATGAGGT AATTTGCTATCTTTGC TGA GGC GAC CGG 2 A C C T 2 T G G A 2 TGA GAT GAT GAT 2 AGG GAAGTA GAA 2 A T T A 2 T T T T 2 A G G G 2 G T T T 2 AT AT AT GA 2 GGA GGACGC GGA 2 C C G A 3 TTAGT A GT G TA A GT CC TTA A GT CG TTACTGGT 3 TGCTAG ACTTA T G T TTAG G T TTA T 3 C T C G CCTA C T C G CC

A 3 C C T C 4 GA GA AG GA 4 G G A A 4 T T T G 4 G G A G 4 T T T C 4 C TT T 4 G C AA ACC A G C AA G C A G 4 C C T C 4 G G A G 4 ACAAAG ACAAAGACAAAG ACAAAG 4 GGCA T GCA T G T A GGCA 5 G G A G 5 C 5 GCTCTC GCTCTC GT TCTC GCTCTC 6 TC ACG AGA AGA GCCAG C G 6 G GACTGC AG TTCGCT AG TTCGCTAG 6 C G C A GGT TG CGGTT T AGCTTA T AGCTTA 6 TG A C GT T C T GC T 6TGTC TGTC TGTC −GT T 7 C C T T 7 C T T T 7 GG AC AC GG 7 A A G A 7 CC GTCC GT 7 CTTT CT C T CTTT −TTT 8 T C G G CGAG CAAG CGAG 8 G A G G 8 TG TGTG TG 8 T T T T 8 C GG TC G TT G TC G 8 T T T T 8 A A A A 9 TGG TGG TGGTGG 9 CCA CCA CCA CCA 9 CC CC CC CC 9 GG AA GG GG 9 AGA G G G AGA AGA

TABLE 15 Listing of sequences with highest sequence identity to the 16SrDNA of MPHPW-1 identified in a BLAST search Percent AlignmentIdentified Accession (Seq ID) Identity Length Organism FJ866783 (91)99.79 1423 Shewanella algae strain PSB-05 HQ851081 (92) 99.79 1423Rhodobacter capsulatus** strain NBY31 US20100044304 99.79 1423Shewanella algae strain SEQ ID 3 (26) IBI-6P IPOD No. FERM BP-10568FJ866781(93) 99.72 1423 Shewanella algae strain PSB-04 GU223381 (94)99.72 1423 Shewanella sp EM0501 HM016084 (95) 99.72 1423 Shewanella spKJW27 X81621 (25) 99.72 1423 Shewanella algae BrY **Apparentlymisclassified as Rhodobacter capsulatus

Example 27 Riboprint® Analysis of MPHPW-1

To determine whether the 16S rDNA genomic region of Shewanella algaestrain MPHPW-1 contained additional distinguishing elements fromShewanella algae BrY, other Shewanella algae strains and a Shewanellachilikensis strain, several genomic DNA preparations of this strain andsix other Shewanella algae or algae-like strains were analyzed byRiboprinter®. These Riboprints® were compared to 7525 patterns containedwithin DuPont Environmental Services and Qualicon libraries compiledfrom samples taken from DuPont as well as another 6950 patterns thatDuPont Qualicon has supplied from standard identified organisms. Basedon the analyses of Riboprint® Batch 1074 (FIG. 25), which provides achromosomal fingerprint of 16S rDNA loci of the tested strains, it isclear that the Riboprint® pattern for strain MPHPW-1 (Sample 1) isunique when compared against the six other strains assayed. The MPHPW-1Riboprint® pattern was also unique among the available DuPont Riboprint®Libraries. The MPHPW-1 pattern was designated as RiboGroup® Identifier212-1074-S-1. It is probable for various strains to share single similarRiboprint® bands generated by hybridizing the labeled E. coli rDNAoperon probe to each strain's genomic Eco RI fragments, but it is theoverall Riboprint® banding pattern that constitutes identification of agiven strain in a specific Riboprint® or Ribogroup® identifier.

This analysis confirmed that the genomic sequences surrounding the rDNAoperons in strain MPHPW-1 have different genomic structure than those inthe Riboprint® database. Shewanella algae strain BrY, whose sequenceidentity to strain MPHPW-1 is 99.72% has a Riboprint® pattern that issimilar to that of MPHPW-1, but its pattern is missing the 1 kb band(fragment). The region for this band is circled in FIG. 25 todemonstrate its absence in row 6, which contains the Riboprint® patternfor Shewanella algae strain BrY. Therefore strain MPHPW-1's Riboprint®is a unique genomic identifier, indicating that MPHPW-1 is a newlyidentified strain.

What is claimed is:
 1. A method for altering the wettability of ahydrocarbon coated surface comprising: a) providing a hydrocarbon-coatedsurface; b) providing a medium selected from the group consisting of: i)a cell-containing medium comprising one or more Shewanella sp.; and ii)a conditioned medium which is substantially cell free and which has beenin contact with one or more Shewanella sp.; wherein the Shewanella sp.comprises a 16S rDNA comprising SEQ ID NO:21, SEQ ID NO:23 and SEQ IDNO:28; and c) contacting said hydrocarbon-coated surface with the mediumof b) wherein the medium alters the wettability of saidhydrocarbon-coated surface.
 2. The method of claim 1 wherein theShewanella sp. further comprises a Riboprint® pattern identifier of212-1074-S-1 as illustrated in FIG.
 25. 3. The method of claim 2 whereinthe Shewanella sp. is MPHPW-1 (ATCC No. PTA-11920).
 4. The method ofclaim 1 wherein the hydrocarbon-coated surface is a component of aterrestrial surface formation or subsurface formation comprisingelements selected from the group consisting of rock, soil, brine, sand,shale, clays and mixtures thereof.
 5. The method of claim 4 wherein thesubsurface formation is an oil well site comprising at least oneproduction well and optionally at least one injection well.
 6. Themethod of claim 4 wherein contacting is achieved by pumping the mediuminto the production well or the injector well.
 7. The method of claim 1wherein the medium further comprises additional components selected fromthe group consisting of at least one electron acceptor, at least oneadditional carbon source, and mixtures thereof.
 8. The method of claim 1wherein the medium further comprises at least one additionalmicroorganism.
 9. A method of treating an environmental site comprising:a) providing an environmental site comprising hydrocarbon-coatedsurfaces; b) contacting the environmental site with the medium of claim1 wherein the hydrocarbon is released from the site; c) collecting waterof the medium and the released hydrocarbon of (b); d) separating thehydrocarbon and water; and e) making medium of claim 1 using the waterof (d) for use in (b).